Process for producing Cis-3-hydroxy-L-proline

ABSTRACT

An isolated gene encodes for a L-proline-3-hydroxylase having the amino acid sequence of SEQ ID NOS: 1, 2, 15, 16 and 17, or for a protein having enzymatic activity to hydroxylate the 3-position of L-proline and to act on free L-proline in the presence of 2-ketoglutaric acid and divalent iron ions to produce cis-3-hydroxy-L-proline. The gene is inserted into a vector and the resulting recombinant DNA is used to create a transformant. L-proline-3-hydroxylase is produced by culturing the transformant in a medium.

[0001] This application is a continuation-in-part of Ser. No.08/474,135, filed Jul. 6, 1995, which is continuation-in-part of Ser.No. 08/301,654 filed Sep. 7, 1994.

FIELD OF THE INVENTION

[0002] The present invention relates to a process for producingcis-3-hydroxy-L-proline. Cis-3-hydroxy-L-proline is useful as a startingcompound for medicines and an additive to foods. The present inventionalso relates to a novel enzyme capable of catalyzing the hydroxylationof L-proline at the 3-position of L-proline (hereinafter referred to as“L-proline-3-hydroxylase”). The novel enzyme is used in theabove-mentioned process.

[0003] The present invention also relates to a gene encoding a proteinhaving an activity of L-proline-3-hydroxylase (hereinafter referred toas “L-proline-3-hydroxylase gene”) which is useful for theabove-mentioned process, a transformant containing the gene, and aprocess for producing L-proline-3-hydroxylase using the transformant.

BACKGROUND OF THE INVENTION

[0004] Heretofore, chemosynthetic methods of producingcis-3-hydroxy-L-proline are known [J. Amer. Chem. Soc., 84, 3980 (1962);J. Amer. Chem. Soc., 85, 2824 (1963); Nature 289, 310 (1981); J. Org.Chem., 54, 1866 (1989); Acta Chemica Scandinavica, 43, 290 (1989)].

[0005] The conventional chemosynthetic methods for producingcis-3-hydroxy-L-proline are not satisfactory for industrial production,because of (1) the expensive raw materials, (2) too many the reactionsteps, (3) the complicated procedures for isolating and purifying theproduct and/or (4) the lower productivity of cis-3-hydroxy-L-proline.

[0006] No chemosynthetic or biological method of producingcis-3-hydroxy-L-proline by hydroxylating L-proline bothregio-selectively and stereo-selectively, had been reported yet.

[0007] The object of the present invention is to provide an advantageousprocess for the production of cis-3-hydroxy-L-proline on theindustrially applicable basis, and the second object of the presentinvention is to provide a novel enzyme which catalyzes the hydroxylationof L-proline at the 3-position of L-proline and which is useful in theabove process.

SUMMARY OF THE INVENTION

[0008] The present invention provides a process for producingcis-3-hydroxy-L-proline, which comprises allowing L-proline to coexitwith 2-ketoglutaric acid, a divalent iron ion and an enzyme source whichcatalyzes the hydroxylation of L-proline at the 3-position of L-prolinein an aqueous medium to convert L-proline into cis-3-hydroxy-L-proline,and recovering the cis-3-hydroxy-L-proline from the aqueous medium.

[0009] The present invention further provides a novel hydroxylase(L-proline-3-hydroxylase) having the following physicochemicalproperties:

[0010] (1) Action and Substrate Specificity:

[0011] The enzyme catalyzes the hydroxylation of L-proline at the3-position of L-proline in the presence of 2-ketoglutaric acid and adivalent iron ion to produce cis-3-hydroxy-L-proline.

[0012] (2) Optimum pH Range:

[0013] The enzyme has an optimum pH range of 6.5 to 7.5, for itsreaction at 30° C. for 20 minutes.

[0014] (3) Stable pH Range:

[0015] The enzyme is stable at pH values of 6.5 to 8.0, when it isallowed to stand at 4° C. for 23 hours.

[0016] (4) Optimum Temperature Range:

[0017] The optimum temperature range is 35 to 40° C. when it is allowedto stand at pH 7.0 for 15 minutes.

[0018] (5) Stable Temperature Range:

[0019] The enzyme is inactivated, when it is allowed to stand at pH 7.5and at 50° C. for 30 minutes.

[0020] (6) Inhibitors:

[0021] The activity of the enzyme is inhibited by metal ions of Zn⁺⁺,Cu⁺⁺, Co⁺⁺ and Ba⁺⁺ and ethylenediaminetetraacetic acid (EDTA).

[0022] (7) Activation:

[0023] The enzyme does not need any cofactor for its activation.L-Ascorbic acid accelerates the activity of the enzyme.

[0024] (8) Km Value:

[0025] The Km value is 0.49 mM for L-proline and is 0.11 mM for2-ketoglutaric acid, when determined in a 100 mMN-tris(hydroxymethyl)methyl-2-aminoethansulfonic acid (TES) buffer (pH7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and apre-determined amount of this enzyme.

[0026] (9) Isoelectric Point;

[0027] The enzyme has an isoelectric point of 4.3 by Phast system.

[0028] (10) Molecular Weight:

[0029] The enzyme has a molecular weight of 35,000±5,000 daltons bysodium dodecylsulfate-polyacrylamide gel electrophoresis.

[0030] (11) N-terminal Amino Acid Sequence:

[0031] The enzyme has an N-terminal amino acid sequence illustrated bySequence No. 5. Sequence No. 5: (N-terminal) 1MetArgSerHisIleLeuGlyArgIleGlu 11 LeuAspGlnGluArgLeuGlyArgAspLeu 21GluTyrLeuAlaThrValProThrVal

[0032] The present invention provides an L-proline-3-hydroxylase geneand a transformant containing the gene for the purpose of producingcis-3-hydroxy-L-proline efficiently and industrially usingL-proline-3-hydroxylase from L-proline that is available at low cost, aprocess for mass-producing the L-proline-3-hydroxylase using thetransformant containing the gene, and a process for producingcis-3-hydroxy-L-proline industrially at low cost using the transformantcontaining the gene conding for L-proline-3-hydroxylase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows the steps of constructing plasmid pTH30 and plasmidpTH40.

[0034] In this, the thick, solid black lines each indicate a clonedStreptomyces sp. TH1 chromosome site. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmids are shown.

[0035]FIG. 2 shows plasmid pTH71 and plasmid pTH75.

[0036] In this, the thick, solid black lines each indicate a clonedStreptomyces sp. TH1 chromosome site. At the part of the thick, solidblack line as drawn along with an arrow, the site has the base sequencecorresponding to Sequence No. 14. The direction of each arrow indicatesthe direction toward the terminal of the sequence of Sequence No. 14from the head thereof. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmids are shown.

[0037]FIG. 3 shows the steps of constructing plasmid pTH50.

[0038] In this, the thick, solid black lines each indicate a partcontaining a Streptomyces sp. TH1 chromosome site-derivedL-proline-3-hydroxylase gene. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmid are shown.

[0039]FIG. 4 shows the steps of constructing plasmid pEX1-5.

[0040] In this, the thick, solid black lines each indicate a partcontaining a Streptomyces sp. TH1 chromosome site-derivedL-proline-3-hydroxylase gene. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmid are shown.

[0041]FIG. 5 shows the steps of constructing plasmid pTH60.

[0042] In this, the thick, solid black lines each indicate a partcontaining a Streptomyces sp. TH1 chromosome site-derivedL-proline-3-hydroxylase gene. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmid are shown.

[0043]FIG. 6 shows the steps of constructing plasmid pTH70.

[0044] In this, the thick, solid black lines each indicate a partcontaining a Streptomyces sp. TH1 chromosome site-derivedL-proline-3-hydroxylase gene. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmid are shown.

[0045]FIG. 7 shows the steps of constructing plasmid pTH80.

[0046] In this, the thick, solid black lines each indicate a partcontaining a Streptomyces sp. TH1 chromosome site-derivedL-proline-3-hydroxylase gene. Ap indicates a pBR322-derivedampicillin-resistant gene. Plac indicates an Escherichia coli lactosepromoter; lacZ indicates a β-galactosidase structural gene; and MCSindicates a multi-cloning site. In this, only the restriction enzymesites having relation to the construction of the plasmid are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0047] As the enzyme source to be used in the process for producingcis-3-hydorxy-L-proline of the present invention, any microorganism canbe used so long as it has an enzymatic activity of catalyzing thehydroxylation of L-proline at the 3-position of L-proline in thepresence of 2-ketoglutaric acid and a divalent iron ion. As themicroorganism having such activity, mention may be made ofmicroorganisms belonging to the genus Streptomyces or Bacillus. Thepreferred strain of such microorganism includes Streptomyces canus ATCC12647, Streptomyces canus ATCC 12646, Streptomyces sp. TH1, and Bacillussp. TH2 and TH3. Specifically, a culture, cells or processed cells ofthese strains can be used. Further, a crude enzyme preparation fromcells of the microorganism as mentioned above, a purified product ofsuch enzyme preparation, an immobilized enzyme preparation, etc. can beused.

[0048]Streptomyces sp. TH1 was newly isolated by the present inventorsfrom the soil. The bacteriological properties of the strain TH1 ismentioned below.

[0049] 1. Morphological Properties:

[0050] The morphological properties when the strain was cultivated onvarious media at 28° C. for 14 days are shown in Table 1 below. TABLE 1Morphological Properties 1) Hyphae Formation of aerial hyphae: YesFragmentation and motility Not observed of aerial hyphae: Fragmentationand motility Not observed of substrate hyphae: 2) Spores Sporulation andPositions Adhered to to which spores adhere: aerial hyphae Formation ofsparangia: Not observed Number of spores on its More than 10 aerialmycelium: Appearance of spore chain: Spiral Characteristics of sporesSpiny Surface structure: Shape and size: Ellipsoidal, 0.5˜0.7 × 1.0˜1.2μm Motility and fragella: Not observed 3) Others Chlamydospores: Notobserved Synnemata: Not observed Pseudosporangia: Not observed Branchingmode of hyphae: Simple branching

[0051] 2. Cultural Characteristics in Various Media:

[0052] The strain TH1 grows normally or vigorously on usual syntheticand natural media, while its substrate hyphae are pale pink (rose),orange or greenish brown. On some media, the strain often produces brownor blackish soluble pigments.

[0053] The cultural characteristics of the strain TH1, when the strainwas cultivated on various media at 28° C. for 14 days, are shown inTable 2. The designation of the colors has been made, according to theclassification of colors indicated in Color Harmony Manual published byContainer Corporation of America. TABLE 2 Cultural Characteristics inVarious Media 1) Sucrose-nitrate Growth: Moderate   agar Color ofsubstrate Pearl pink (3 ca) hyphae; Adhesion of None aerial hyphae:Soluble pigments: None 2) Glucose- Growth: Moderate   asparagine agarColor of substrate Pearl pink (3 ca) hyphae; Adhesion of None aerialhyphae: Soluble pigments: None 3) Glycerol- Growth: Moderate  asparagine agar Color of substrate Pearl pink˜bright hyphae; orange(3ca˜4na) Adhesion and color Poor, white (a) of aerial hyphae: Solublepigments: Produced only a little (Pale yellow) 4) Inorganic salts-Growth: Moderate   starch agar Color of substrate Pearl (2 ba) hyphae:Adhesion and color Moderate, white (a) of aerial hyphae: Solublepigments: Negative 5) Tyrosine agar Growth: Moderate Color of substrateLight olive (1 1/2 ie) hyphae: Adhesion and color Moderate, of aerialhyphae: white˜olive gray Soluble pigments: Produced (black) 6) Nutrientagar Growth: Poor Color of substrate Light melon hyphae: yellow (3 ea)Adhesion of None aerial hyphae; Soluble pigments: None 7) Yeast extract-Growth: Abundant   malt extract agar Color of substrate Flesh pinkhyphae: (4 ca) Adhesion and color of Moderate, aerial hyphae: white (a)Soluble pigments: Produced only a little (ocher) 8) Oatmeal agar Growth;Poor Color of substrate Yellow tint hyphae: (1 ba) Adhesion and color ofModerate, aerial hyphae: white (a) Soluble pigments: None

[0054] 3. Physiological Properties:

[0055] The physiological properties of the strain TH1 is shown in Table3, in which the “temperature range for growth” indicates the results ofthe strain after 7 day-cultivation by shaking. The remaining itemsindicate the results after 2 to 3 week-cultivation at 28° C. TABLE 3Physiological Properties 1) Temperature Range for Growth 23 to 37° C. 2)Liquefaction of Gelatin No 3) Hydrolysis of Starch Yes 4) Coagulation ofSkim Milk Powder No 5) Peptonization of Skim Milk Powder No 6) Formationof Melanoid Pigments (1) Peptone-yeast extract-iron agar Yes (2)Tyrosine agar Yes 7) Utilization of Carbon Sources(*) L-Arabinose +D-Glucose + D-Xylose + Sucrose + Raffinose + D-Fructose + Rhamnose −Inositol + D-Mannitol +

[0056] 4. Chemotaxonomic Properties:

[0057] The configuration of diaminopimelic acid in the cells is LL type.Accordingly, the strain TH1 is classified to the genus Streptomyces ofactinomycetes in view of its morphological properties that it formsspiral spore chain consisting of more than ten spores on its aerialmycelium, and in view of its chemotaxonomic properties that thediaminopimelic acid contained in its cell wall is L,L-diaminopimelicacid (type I). The strain TH1 was identified as Streptomyces sp. TH1,and the strain has been deposited with National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology inJapan as of Sep. 1, 1993 under FERM BP-4399 in terms of the BudapestTreaty.

[0058] Bacillus sp. TH2 and TH3 were newly isolated by the presentinventors from the soil. The bacteriological properties of the strainsTH2 and TH3 are mentioned below.

[0059] Both the strains TH2 and TH3 are the same in the followingproperties except that the cell sizes of the strain TH2 and TH3 are 1.5to 1.8×3 to 4 μm and 1.2 to 1.5×3.5 to 4 μm, respectively, and the basecompositions of DNA (G+C mol %) of the strains TH2 and TH3 are 36.46%and 37.74%, respectively.

[0060] 1. Morphological Properties:

[0061] The strains had the morphological properties shown in Table 4below. TABLE 4 Morphological Properties 1) Cells Morphology RodPolymorphism Not observed Motility Not observed (Position of flagella)2) Endo Sporulation Observed   Spores Morphology Ellipse Positions Theend position

[0062] 2. Cultural Characteristics in Various Media:

[0063] The Cultural characteristics of the strains TH2 and TH3 are shownin Table 5 below. TABLE 5 Cultural Characteristics in Various MediaBouillon-Agar Growth Abundant Medium Shape of surface Smooth (Meatextract) Color Pale yellow white Gloss None Diffusible pigments NegativeBouillon-liquid Growth appearance Grown only a Medium Turbidity littleon surface (Meat extract) of the medium Positive Bouillon- Liquefactionof Positive Gelatin-medium gelatin Litmus milk Reaction Acid CoagulationNegative Liquefaction Positive

[0064] 3. Physiological Properties:

[0065] The physiological properties of the strains TH2 and TH3 are shownin Table 6. TABLE 6 (1) Physiological Properties (1) Gram staining + (2)Reduction to nitrite + (3) Denitrification + reaction (4) Methyl redtest − (5) VP test − (6) Indole production − (7) Hydrogen sulfideproduction (8) Hydrolysis of starch − (9) Utilization of + citric acidKoser's method − Christensen's method W (10) Utilization of inorganicnitrogen Nitrates − Ammonium salts + (11) Pigment production King Amedium − King B medium − (12) Urease − (13) Oxidase − (14) Catalase +(15) Growth range 5 to 9.7 pH; Optimum PH; 7.1 Temperature; 4 to 36° C.Optimum temp; around 33° C. (16) Attitude towards oxygen Aerobic +Anaerobic + (facultative anaerobic) (17) OF test oxidation

[0066] TABLE 6 (2) Production of Acid and Gas from Carbohydrates AcidGas production production L-Arabinose − − D-Xylose − − D-Glucose + −D-Mannose − − D-Fructose + − D-Galactose − − Maltose + − Sucrose + −Lactose − − Trehalose + − D-Sorbitol − − D-Mannitol − − Inositol − −Glycerol − − Starch + −

[0067] 4. The other Properties and the Chemotaxonomic Properties:

[0068] The other properties of the strains TH2 and TH3 are shown inTable 7, and the chemotaxonomic properties are shown in Table 8 below.TABLE 7 Other Properties Degradation of Esculin + Degradation of Malonicacid − Degradation of Arginine + Decarboxylation of Lysine −Decarboxylation of Ornithine − Deamination of Phenylalanine − Resistanceto NaCl (7.5%) − Phosphatase +

[0069] TABLE 8 Chemotaxonomic Properties 1) Cellular lipids UbiquinoneNegative Menaquinone MK-7 2) Diamino acid composition of meso-A₂ pm cellwall peptidoglycan

[0070] The strains having the bacteriological properties mentioned abovewere compared with the strains indicated in Bergey's Manual ofSystematic Bacteriology (Vol. 2, 1986).

[0071] Both the strains TH2 and TH3 are classified to the genus Bacillusof bacteria. The strains TH2 and TH3 were identified as Bacillus sp. TH2and Bacillus sp. TH3, respectively. These strains have been depositedwith the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology in Japan as of Sep. 1, 1993 underFERM BP-4397 for the strain TH2 and under FERM BP-4398 for the strainTH3, both in terms of the Budapest Treaty.

[0072] The medium for cultivating these microorganisms may be any ofnatural media and synthetic media, so long as it contains carbonsources, nitrogen sources, inorganic salts, etc. that may be assimilatedby microorganisms having an activity of catalyzing the hydroxylation ofL-proline to produce cis-3-hydroxy-L-proline.

[0073] As the carbon sources, carbohydrates such as glucose, fructose,sucrose, molasses containing these compounds, starch and starchhydrolysates, etc.; organic acids such as acetic acid, propionic acid,etc.; alcohols such as ethanol, propanol, etc. may be used.

[0074] As the nitrogen sources, ammonia; ammonium salts of variousinorganic and organic acids such as ammonium chloride, ammonium sulfate,ammonium acetate, and ammonium phosphate, etc.; othernitrogen-containing compounds; peptone, meat extracts, yeast extracts,corn steep liquor, casein hydrolysates, soy bean cakes, soy bean cakehydrolysates, various cultured cells of microorganisms, their digestedproducts, etc. may be used.

[0075] The inorganic material includes, for example, potassiumdihydrogen phosphate, dipotassium hydrogen phosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, copper sulfate, and calcium carbonate, etc.

[0076] The cultivation of these microorganisms is carried out underaerobic conditions, for example, by shaking culture or bysubmerged-aerial stirring culture. The temperature for the cultivationis preferably from 15 to 37° C., and the period for the cultivation isgenerally from 16 to 96 hours. During the cultivation, the pH of themedium is kept at 5.0 to 9.0 with inorganic or organic acids, alkalinesolutions, urea, calcium carbonate, ammonia, etc.

[0077] The thus-cultivated microorganisms can be used as the enzymesoured to be used in the process for producing cis-3-hydroxy-L-proline.The amount of the enzyme source to be used in the process for producingcis-3-hydroxy-L-proline depends on the amount of the substrate to beused in the process. Usually, it may be from 1.0 to 10,000,000 U,preferably from 1,000 to 3,000,000 U/liter of the aqueous medium.

[0078] In the case of using microbial cells and products obtained byprocessing microbial cells, the concentration of wet cells to be usedmay be generally from 1 to 300 g/l.

[0079] The enzyme activity for producing one nmol ofcis-3-hydroxy-L-proline for one minute under the conditions mentionedbelow is defined as one unit (U).

[0080] The enzyme preparation to be determined is added to 100 mM TESbuffer (pH 7.0) containing 5 mM L-proline, 5 mM 2-ketoglutaric acid, 1mM ferrous sulfate and 5 mM L-ascorbic acid to make 100 μl in total, andthe mixture was allowed to stand at 35° C. for 10 minutes. The reactionmixture is heated at 100° C. for 2 minutes so as to stop the reaction,and the amount of cis-3-hydroxy-L-proline produced in the reactionmixture is determined by high performance liquid chromatography(hereinafter referred to as HPLC).

[0081] For the determination, any method capable of determining theamount of cis-3-hydroxy-L-proline may be employed. For instance,generally usable are a post-column derivatization method where HPLC isutilized, and a pre-column derivatization method where the compound tobe determined in the reaction mixture is previously reacted with7-chloro-4-nitrobenz-2-oxa-1,3-diazole (hereinafter referred to as NBD)to form its NBD-derivative, the derivative is separated byreversed-phase chromatography using HPLC and the thus-separatedderivative is quantitatively determined by spectrofluorometry(excitation wavelength: 503 nm, emission wavelength: 541 nm). Thepre-column derivatization method may be conducted, according to themethod of William J. Lindblad & Robert F. Diegelmann, et al. [seeAnalytical Biochemistry, 138, 390-395 (1984)].

[0082] The concentration of L-proline to be used in the process forproducing cis-3-hydroxy-L-proline may be from 1 mM to 2M.

[0083] The process for producing cis-3-hydroxy-L-proline needs adivalent iron ion. The concentration of the divalent iron ion maygenerally be from 1 to 100 mM. Any divalent iron ion may be used so longas it does not inhibit the enzyme reaction. For instance, sulfides suchas ferrous sulfate, chlorides such as ferrous chloride, ferrouscarbonate, the salts of organic acids such as citrates, lactates,fumarates may be used.

[0084] The process also needs 2-ketoglutaric acid. The concentration of2-ketoglutaric acid is generally from 1 mM to 2M. 2-Ketoglutaric aciditself may be added to the aqueous medium, or alternatively, thecompound that may be converted into 2-ketoglutaric acid by the metabolicactivity of the microorganism used in the enzymatic reaction may beadded thereto. The compound includes, for example, saccharides such asglucose, glutamic acid and succinic acid. These compounds may be usedsingly or in combination.

[0085] The aqueous medium to be used in the process for producingcis-3-hydroxy-L-proline includes, for example, water, buffers such asphosphates, carbonates, acetates, borates, citrates, tris-buffers,alcohols such as methanol and ethanol, esters such as ethyl acetate,ketones such as acetone, and amides such as acetamide.

[0086] The enzymatic reaction may be carried out in the culture mediumwhere the above-mentioned microorganisms having an activity ofcatalyzing hydroxylation of L-proline to produce cis-3-hydroxy-L-prolineare being cultivated or have been cultivated, or alternatively, theenzymatic reaction may also be carried out in an aqueous mediumcontaining the cells of the above-mentioned microorganisms separatedfrom the culture, processed cells, or a purified or crude enzyme derivedfrom the cells.

[0087] Processed cells of the microorganisms include, for example, driedcells, lyophilized cells, surfactant-treated cells,enzymatically-treated cells, ultrasonically-treated cells,mechanically-ground cells, mechanically-compressed cells,solvent-treated cells, fractionated cell proteins, immobilized cells,immobilized materials obtained by processing their cells, etc.

[0088] The enzymatic reaction is generally carried out at a temperatureof 15 to 50° C. and at a pH of 6.0 to 9.0, for a period of 1 to 96hours. If desired, surfactants and/or organic solvents may be addedduring the processing of the cells or during the enzymatic reaction.

[0089] As the surfactants, mention may be made of cationic surfactantssuch as polyoxyethylene-stearylamine (e.g., Nymeen S-215, produced byNippon Oils and Fats Co.), cetyltrimethylammonium bromide, Cation FB andCation F2-40E, etc.; anionic surfactants such as sodiumoleylamidosulfate, Newrex TAB and Rapizble 80; ampholytic surfactantssuch as polyoxyethylene-sorbitan monostearate (e.g., Nonion ST221);other tertiary amines PB, hexadecyldimethylamine, etc. Any surfactantthat may promote the reaction may be employed. The concentration of thesurfactant to be employed in the reaction may be generally from 0.1 to50 mg/ml, preferably from 1 to 20 mg/ml.

[0090] As the organic solvent, mention may be made of toluene, xylene,aliphatic alcohols, benzene, ethyl acetates etc. Generally, theconcentration of the solvent in the process may be from 0.1 to 50 μl/ml,preferably from 1 to 20 μl/ml.

[0091] To recover cis-3-hydroxy-L-proline from the aqueous medium,ordinary separation methods such as column chromatography usingion-exchange resins, crystallization, etc. may be employed. Thestructure of the recovered cis-3-hydroxy-L-proline may be identified byordinary analytical methods such as ¹³C-NMR spectrum ¹H-NMR spectrum,mass spectrum, specific rotation or the like.

[0092] Next, the novel enzyme, the L-proline-3-hydroxylase of thepresent invention is described below.

[0093] The L-proline-3-hydroxylase may be obtained by cultivating amicroorganism having an ability to produce L-proline-3-hydroxylase in amedium so as to produce and accumulate the L-proline-3-hydroxylase inthe culture medium, and recovering the L-proline-3-hydroxylase from theculture.

[0094] Any microorganisms having an ability to produceL-proline-3-hydroxylase may be employed. For example, microorganismsbelonging to the genus Streptomyces or Bacillus and having such activitycan be used. Specific examples are Streptomyces canus ATCC12647,Streptomyces canus ATCC12646, Streptomyces sp. TH1, Bacillus sp. TH2 andBacillus sp. TH3, a sub-cultivated strain thereof, its mutant thereof,its derivative thereof, etc.

[0095] The medium for cultivating these microorganisms may be any ofnatural media and synthetic media, so long as it contains carbonsources, nitrogen sources, inorganic salts, etc. that may be assimilatedby microorganisms having an ability to produce L-proline-4-hydroxylase.

[0096] The carbon source includes, for example, carbohydrates such asglucose, fructose, sucrose, molasses containing these components, starchand starch hydrolysates; organic acids such as acetic acid and propionicacid; alcohols such as ethanol and propanol which may be assimilated bythe microorganisms.

[0097] As the nitrogen source, ammonia; ammonium salts of variousinorganic acids and organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate and ammonium phosphate; othernitrogen-containing compounds; peptone, meat extracts, yeast extracts,corn steep liquor, casein hydrolysates, soy bean cakes, soy bean cakehydrolysates, various microorganisms for fermentation, their digestedproducts, etc. may be used.

[0098] As the inorganic material, dipotassium hydrogen phosphate,potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,calcium carbonate, etc. can be used.

[0099] The cultivation of these microorganisms is carried out underaerobic conditions, for example, with shaking culture orsubmerged-aerial stirring culture. The temperature for the cultivationis preferably from 15 to 37° C., and the period for the cultivation isgenerally from 16 to 96 hours.

[0100] During the cultivation, the pH of the medium is kept at 5.0 to9.0 with inorganic or organic acids, alkaline solutions, urea, calciumcarbonate, ammonia, etc. During the cultivation, L-proline may be added,if desired.

[0101] To isolate and purify the enzyme from the culture containing theenzyme, any ordinary method for isolating and purifying an enzyme may beemployed. For instance, the culture is subjected to centrifugation tocollect the cultivated cells therefrom, and the cells are fully washedand then disrupted by an ultrasonic cell disrupter, a French press, aManton-Gauline homogenizer, a Dyno mill, etc. to obtain a cell-freeextract. The cell-free extract is again subjected to centrifugation, andthe enzyme in the resulting supernatant is then purified, for example,by salting-out with ammonium sulfate or the like, by anion-exchangechromatography with diethylaminoethyl (DEAE)-Sepharose or the like, byhydrophobic chromatography with butyl-Sepharose, phenyl-Sepharose or thelike, by dye affinity chromatography with red-Agarose or the like, bygel filtration with molecular sieves or by electrophoresis such asisoelectric point electrophoresis or the like. In this way, a pureproduct of the enzyme is obtained. The activity of theL-proline-3-hydroxylase thus isolated may be determined by the samemethod as mentioned above.

[0102] The L-proline-3-hydroxylase, thus obtained according to themanner mentioned above, has the following physicochemical properties (1)to (11):

[0103] (1) Action and Substrate Specificity:

[0104] The enzyme catalyzes the hydroxylation of L-proline at the3-position of L-proline in the presence of 2-ketoglutaric acid and adivalent iron ion to produce cis-3-hydroxy-L-proline.

[0105] (2) Optimum pH Range:

[0106] The enzyme has an optimum pH range of 6.5 to 7.5 for its reactionat 30° C. for 20 minutes.

[0107] (3) Stable pH Range:

[0108] The enzyme is stable at pH values ranging from 6.5 to 8.0, whenit is allowed to stand at 4° C. for 23 hours.

[0109] (4) Optimum Temperature Range:

[0110] The optimum temperature range is in 35 to 40° C., when it isallowed to stand at pH 7.0 for 15 minutes.

[0111] (5) Stable Temperature Range:

[0112] The enzyme is inactivated, when it is allowed to stand at pH 7.5and at 50° C. for 30 minutes.

[0113] (6) Inhibitors:

[0114] The activity of the enzyme is inhibited by metal ions of Zn⁺⁺,Cu⁺⁺, Co⁺⁺ and Ba⁺⁺ and ethylenediaminetetraacetic acid (EDTA).

[0115] (7) Activation:

[0116] The enzyme does not need any cofactor for its activation.L-Ascorbic acid accelerates the activity of the enzyme.

[0117] (8) Km Value:

[0118] The Km value is 0.49 mM for L-proline and is 0.11 mM for2-ketoglutaric acid, when determined in a 100 mMN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer(pH7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and apre-determined amount of this enzyme.

[0119] (9) Isoelectric Point:

[0120] The enzyme has an isoelectric point of 4.3 by Phast system.

[0121] (10) Molecular Weight:

[0122] The enzyme has a molecular weight of 35,000+5,000 daltons bysodium dodecylsulfate-polyacrylamide gel electrophoresis.

[0123] (11) N-terminal Amino Acid Sequence;

[0124] The enzyme has an N-terminal amino acid sequence illustrated bySequence No. 5. Sequence No. 5: (N-terminal) 1MetArgSerHisIleLeuGlyArgIleGlu 11 LeuAspGlnGluArgLeuGlyArgAspLeu 21GluTyrLeuAlaThrValProThrVal

[0125] The L-proline-3-hydroxylases of the present invention are enzymeswhich hydroxylate free L-proline in the presence of 2-ketoglutaric acidand divalent iron ions to produce cis-3-hydroxy-L-proline.

[0126] The present invention encompasses any and every protein havingthe enzymatic activity of hydroxylating the 3-position of L-proline,which includes, for example, a protein having the amino acid sequenceindicated by Sequence No. 1 or 2, a fused protein having an amino acidsequence that results from the protein or a protein having a partialamino acid sequence of the protein as bonded to a peptide having apartial amino acid sequence of an Escherichia coli-derivedβ-galactosidase protein, a fused protein having an amino acid sequencethat results from the protein having the amino acid sequence indicatedby Sequence No. 1 or 2 or a protein having a partial amino acid sequenceof the protein as bonded to a peptide having a partial amino acidsequence of an E. coli-derived maltose-binding protein, etc. Examples ofthe fused proteins include proteins having the amino acid sequence asindicated by Sequence No. 15, 16 or 17, etc.

[0127] The amino acid sequence indicated by Sequence No. 1, 2, 15, 16 or17 includes proteins having an amino acid sequence with one or moreamino acids substituted, deleted or added and having the enzymaticactivity of hydroxylating the 3-position of L-proline. The substitution,the deletion and the addition of amino acids can be conducted inaccordance with the methods described in Nucleic Acids Research, Vol.10, pp. 6487-6500 (1982); Proc. Natl. Acad. Sci., USA, Vol. 79, pp.6409-6413 (1982); Proc. Natl. Acad. Sci., USA, Vol. 81, pp. 5662-5666(1984); Science, Vol. 224, pp. 1431-1433 (1984); PCTWO85/00817 (1985);Nature, Vol. 316, pp. 601-605 (1985); Gene, Vol. 34, pp. 315-323 (1985);Nucleic Acids Research, Vol. 13, pp. 4431-4442 (1985); Current Protocolsin Molecular Biology, Chap. 8, Mutagenesis of Cloned DNA, John Wiley &Sons, Inc. (1989), etc.

[0128] The present invention encompasses any and everyL-proline-3-hydroxylase genes of a DNA fragment containing a gene thatcodes for a protein having the enzymatic activity of hydroxylating the3-position of L-proline, and this may include, for example, genes codingfor the protein having the amino acid sequence as indicated by SequenceNo. 1, 2, 15, 16, or 17, and also genes which code for a protein thathas an amino acid sequence corresponding to the amino acid sequence asindicated by Sequence No. 1, 2, 15, 16 or 17 and derived therefrom bysubstitution, deletion or addition of at least one amino acid and whichhave the enzymatic activity of hydroxylating the 3-position ofL-proline. Concretely mentioned are DNAs indicated by Sequence Nos. 3,4, 13 and 14.

[0129] The L-proline-3-hydroxylases genes of the present inventioninclude the DNAs as defined hereinabove and also DNAs as derivedtherefrom by mutation, such as substituting mutation, deleting mutation,inserting mutation or the like, to be conducted to the extent that themutated DNAs do not lose the L-proline-3-hydroxylases activity, forexample, DNAs with homology to Sequence No. 3, 4, 13 or 14. Suchhomologous DNAs are those to be obtained by colony hybridization orplaque hybridization using, as a probe, the DNA having the nucleotidesequence as indicated by Sequence No. 3, 4, 13 or 14. These treatmentscan be conducted in accordance with known in vitro recombinationtechniques [see Molecular Cloning: A Laboratory Manual, 2nd Ed., editedby Sambrook, Fritsch, Maniatis, published by Cold Spring HarborLaboratory Press, 1989].

[0130] DNA fragments containing the L-proline-3-hydroxylases gene can beobtained from microorganisms having the ability of hydroxylatingL-proline to produce cis-3-hydroxy-L-proline. As the microorganisms, anymicroorganism having the ability of hydroxylating L-proline to producecis-3-hydroxy-L-proline can be employed in the present invention. Aspreferable example of such a microorganism, microorganisms belonging tothe genus Streptomyces or Bacillus and having the enzymatic activity ofhydroxylating the 3-position of L-proline can be mentioned. Morepreferable examples thereof include Streptomyces canus ATCC12647,Streptomyces canus ATCC12646, Streptomyces sp. TH1 (FERM BP-4399),Bacillus sp. TH2 (FERM BP-4397), Bacillus sp. TH3 (FERM BP-4398), ormutants or derivatives of these strains.

[0131] Methods for obtaining L-proline-3-hydroxylases genes derived frommicroorganisms having the ability of producing L-proline-3-hydroxylasesare described below.

[0132] Chromosomal DNA is prepared from a microorganism having theability of producing L-proline-3-hydroxylase through a usual DNAisolation method, for example, a phenol method [see Biochim. Biophys.Acta, 72, 619-629 (1963)]. The thus-obtained chromosomal DNA is cleavedwith suitable restriction enzymes, then the resulting fragments areinserted into vector DNAs to construct chromosomal DNA libraries for thechromosomes of the microorganisms. Using the chromosomal DNA library,host microorganisms can be transformed. The transformants containing theL-proline-3-hydroxylase gene are selected from the obtainedtransformants by the hybridization method. DNAs containing the intendedgene can be obtained from the thus-selected transformants.

[0133] The process comprising a series of such steps can be conducted inaccordance with known in vitro recombination method (molecular Cloning,A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch andManiatis, Cold Spring Harbor Laboratory Press, 1989).

[0134] As the vector DNAs that are used to construct the chromosomal DNAlibrary of the microorganism having the ability of producingL-proline-3-hydroxylase, phage vectors and plasmid vectors can be usedif they can be replicated autonomously in Escherichia coli K12 strain.Preferable examples of the vector DNA include λ ZAPII, pUC18 andpBluescript (commercially available from STRATAGENE Co.).

[0135] As the host microorganisms that are used to construct thechromosomal DNA library of the microorganism having the ability ofproducing L-proline-3-hydroxylase, any of the microorganisms belongingto the genus Escherichia can be used. Preferable examples of the hostmicroorganisms include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1,E. coli MC1000, etc.

[0136] Based on the information about the amino acid sequence ofL-proline-3-hydroxylase, DNA primers are synthesized. Using the DNAprimers, DNA fragments are prepared through polymerase chain reaction(hereinafter referred to as PCR). Using the thus-obtained DNA fragments,transformants containing an L-proline-3-hydroxylase gene can be selectedby the hybridization method.

[0137] The information on the amino acid sequences ofL-proline-3-hydroxylases can be obtained through analysis of pureL-proline-3-hydroxylases using ordinary amino acid sequencers, such asProtein Sequencer Model PPSQ-10 (produced by Shimadzu Seisakusho K.K.).As the information on the amino acid sequences thus obtained, concretelymentioned are the amino acid sequences as indicated by Sequence Nos. 5to 7, etc.

[0138] The DNA primers can be synthesized by means of an ordinary DNAsynthesizers, for example, 380A•DNA Synthesizer (produced by AppliedBiosystems Co.), etc.

[0139] As the probes for the hybridization, usable are partial fragmentsof L-proline-3-hydroxylases genes, which can be obtained through PCR.For example, a DNA as indicated by Sequence No. 8 in the Sequence List(this corresponds to a sense chain DNA coding for from the first to thesixth amino acids in the amino acid sequence of Sequence No. 1) and aDNA as indicated by Sequence No. 10 in the Sequence List (thiscorresponds to an anti-sense chain DNA coding for from the 21st to 25thamino acids in the amino acid sequence of Sequence No. 1) are chemicallysynthesized Through PCR using these partial fragment as DNA primers,obtained is a DNA fragment of 74 bpas indicated by Sequence No. 11. Thethus-obtained DNA fragment can be used as the probe for thehybridization.

[0140] The DNA which is obtained from the transformant as selectedthrough the hybridization and which contains an L-proline-3-hydroxylasesgene is cleaved with suitable restriction enzymes, such as PstI or thelike, and then cloned into plasmids, such as pBluescript KS(+)(commercially available from STRATAGENE Co.). The nucleotide sequence ofthe above-mentioned gene can be determined by ordinary base sequencingmethods, such as the dideoxy chain termination method of Sanger et al[see Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)]. The determination ofthe nucleotide sequence can be conducted using automatic DNA sequencers,such as 373A•DNA Sequencer (produced by Applied Biosystems Co.) or thelike.

[0141] As the thus-determined nucleotide sequences ofL-proline-3-hydroxylases genes, for example the nucleotide sequences asindicated by Sequence Nos. 3, 4, 13 and 14 can be mentioned.

[0142] The DNA that codes for an L-proline-3-hydroxylase of the presentinvention can be introduced into vectors in a usual manner. As thevectors containing the DNA that codes for an L-proline-3-hydroxylases ofthe present invention, for example, pTH30, pTH71, pTH75, etc. can bementioned. Escherichia coli XL2-Blue/pTH30 which is Escherichia colicontaining pTH30 and Escherichia Coli XL2-Blue/pTH75 which isEscherichia coli containing pTH75 were deposited at the NationalInstitute of Bioscience and Human-Technology of the Agency of IndustrialScience and Technology (which is located at 1-3, Higashi 1-chome,Tsukuba-shi, Ibaragi-ken 305, Japan) as of Mar. 2, 1995 under FERMBP-5026 for the strain XL2-Blue/pTH30 and as of Feb. 22, 1996 under FERMBP-55409 for the strain in XL2-Blue/pTH75, both in terms of the BudapestTreaty.

[0143] To express the thus-obtained L-proline-3-hydroxylase genes inhosts, the DNA fragment containing the L-proline-3-hydroxylase gene isfirst cleaved with a restriction enzyme or deoxyribonucleases (DNases)to form a DNA fragment of a suitable length containing theL-proline-3-hydroxylases gene. The thus-formed DNA fragment is insertedinto an expression vector at the downstream position of the promotertherein, and thereafter the expression vector having the thus-insertedDNA therein is introduced into a host cell suitable for the expressionvector.

[0144] As the host, usable is any one capable of being expressed in theintended gene. For example, the host includes microorganisms belongingto the genus Escherichia, Serratia, Corynebacterium, Brevibacterium,Pseudomonas, Bacillus, etc., as well as yeast strains, animal cellhosts, etc.

[0145] As the expression vector, usable is one which is autonomouslyreplicable in the above-mentioned hosts or capable of being insertedinto their chromosomes and which contains a promoter at the position atwhich the L-proline-3-hydroxylases gene can be transcribed.

[0146] When the microorganisms such as Escherichia coli or the like. areused as the host, it is desirable that the L-proline-3-hydroxylasesexpression vector is replicated autonomously in the microorganisms andis composed of a promoter, a ribosome-bonding sequence, anL-proline-3-hydroxylase gene and a transcription terminator sequence. Aregulatory gene may be contained therein.

[0147] As examples of the expression vector, mentioned are pBTrp2,pBTac1, pBTac2 (all commercially available from Boehringer MannheimCo.); pKYP10 (see Japanese Published Unexamined Patent Application No.58-110600); pKYP200 [see Agric. Biol. Chem., Vol. 48, pp. 669-675(1984)]; pLSA1 [see Agric. Biol. Chem., Vol. 53, p. 277 (1989)]; pGEL1[see Proc. Natl. Acad. Sci., USA, Vol. 82, p. 4306 (1985)]; pBluescript(produced by STRATAGENE Co.); pTrs30 [prepared from Escherichia coliJM109/pTrS30 (FERM BP-5407); pTrs32 [prepared from Escherichia coliJM109/pTrs32 (FERM BP-5408)], etc.

[0148] As the promoter, usable is any one capable of being expressed inhosts such as Escherichia coli, etc. For example, mentioned arepromoters derived from Escherichia coli, phage, etc., such as trppromoter (Ptrp), lac promoter (Plac), PL promoter and PR promoter. Alsousable are artificially designed and modified promoters, such as Ptrpx2to be prepared by connecting two Ptrp's in series, as well as tacpromoter.

[0149] As the ribosome-bonding sequence, any one capable of beingexpressed in hosts such as Escherichia coli. can be used. However, it isdesirable to use plasmids having a ribosome-bonding sequence and aninitiation codon as spaced at suitable intervals therebetween (forexample, by from 6 to 18 bases).

[0150] The L-proline-3-hydroxylase gene may be any and every gene thatcodes for an L-proline-3-hydroxylase. However, it is desirable that thebases constituting the DNA sequence of the gene are suitably substitutedin order that the substituted DNA sequence can be constituted of codonmost suitable for expression in the host microorganisms to be used.

[0151] Transcription terminator sequences are not always necessary forthe expression of the genes of the present invention. However, it isdesirable that a transcription terminator sequence is arranged justafter the structural gene.

[0152] Examples of the hosts usable in the present invention includeEscherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coliDH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coliW1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coliNo. 49, Escherichia coli W3110, Escherichia coli NY49, Bacillussubtilis, Bacillus amyloliquefacines, Brevibacterium immariophilumATCC14068, Brevibacterium saccharolvticum ATCC14066, Brevibacteriumflavum ATCC14067, Brevibacterium lactofermentum ATCC13869,Corynebacterium glutamicum ATCC13032, Corynebacterium acetoacidophilumATCC13870, Microbacterium ammoniaphilum ATCC15354, etc.

[0153] When yeast strains are used as the host, for example, YEp13(ATCC37155), YEp24 (ATCC37051), YCp50 (ATCC37419), etc. can be used asthe expression vector.

[0154] As the promoter, any one that can be expressed in the host ofyeast strains can be used. For example, usable are promoters in genes ofglycolases, such as hexosekinase, etc., as well as other promoters suchas gal 1 promoter, gal 10 promoter, heat-shock protein promoters, MF α1promoter, CUP 1 promoter, etc.

[0155] As examples of the host cells, Saccharomyces cerevisae,Schizosaccharomyces probe, Kluvveromvces lactis, Trichosporon Dullulans,Schwanniomyces alluvius, etc. can be mentioned.

[0156] When animal cells are used as the host, for example, pcDNA I/Amp,pcDNA I, pcDM8 (all commercially available from Funakoshi Co.), etc. canbe used as the expression vector. As the promoter, any one that can beexpressed in the host of animal cells can be used. For example, usableare promoters such as those in human CMV IE (immediate early) genes. Anenhancer of human CMV IE genes can be used along with the promoter.

[0157] As examples of the host cells, Namalwa cells, HBT 5637 cells (seeJapanese Published Unexamined Patent Application No. 299/88), COS cells,CHO cells, etc. can be used.

[0158] To introduce DNA into animal cells, any and every method capableof introducing DNA into animal cells can be employed herein. Forexample, employable are electroporation methods [see Miyaji et al.,Cytotechnology, 3, 133 (1990)], calcium phosphate methods (see JapanesePublished Unexamined Patent Application No. 227075/90), lipofectionmethods [see Philip L. Felgner, et al., Proc. Natl. Acad. Sci., USA, 84,7413 (1987)], etc. The resulting transformants can be collected andcultivated in accordance with the methods described in JapanesePublished Unexamined Patent Application Nos. 227075/90 and 257891/90.

[0159] The transformants obtained in the manner mentioned above can becultivated according to ordinary incubation methods.

[0160] As the media for cultivating the transformants obtained by usingthe hosts of microorganisms such as Escherichia coli, yeast strains,etc., both natural and synthetic media can be employed so long as theyproperly contain carbon sources, nitrogen sources, inorganic salts andother materials capable of being assimilated by the microbial hosts andin which the transformants can be efficiently cultivated.

[0161] Any carbon sources that can be assimilated by the microorganismsmay be used. Examples of the carbon source include carbohydrates such asglucose, fructose, sucrose, molasses containing these components, starchand starch hydrolyzates; organic acids such as acetic acid and propionicacid; and alcohols such as ethanol and propanol.

[0162] As the nitrogen sources, ammonia, ammonium salts of inorganic andorganic acids such as ammonium chloride, ammonium sulfate, ammoniumacetate and ammonium phosphate, other nitrogen-containing compounds,peptone, meat extracts, yeast extracts, corn steep liquor, caseinhydrolyzates, soybean cakes, soybean cake hydrolyzates, culturedfermented cells, their digested products, etc. may be used.

[0163] As inorganic salts, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, copper sulfate, calciumcarbonate, etc. may be used.

[0164] The cultivation is conducted under aerobic conditions, forexample, with shaking culture or submerged-aerial stirring culture. Thetemperature for the cultivation is 15 to 40° C. The period for thecultivation is usually 16 to 96 hours. During the cultivation, the pH ofthe medium is kept at 3.0 to 9.0. The pH is adjusted using inorganic ororganic acids, alkaline solutions, urea, calcium carbonate, ammonia orthe like.

[0165] L-Proline is suitably added to the media in such a manner thatits concentration may be from 5 to 1000 mM, preferably from 20 to 200mM, whereby the intended L-proline-3-hydroxylases can be produced moreefficiently.

[0166] Antibiotics such as ampicillin, tetracycline or the like may beadded to the medium during the cultivation, if required.

[0167] For the cultivation of the microorganisms which are transformedwith the expression vector using the inducible promoter, inducers may beadded to the medium, if required. For example, in cultivation ofmicroorganisms transformed with the expression vector using lacpromoter, isopropyl-β-D-thiogalactopyraNos.ide (IPTG) maybe added to themedium. In cultivation of microorganisms transformed with the expressionvector using trp promoter, indoleacrylic acid (IAA) may be added to themedium.

[0168] As the medium for cultivating the transformants which areobtained by using the animal cells as a host cell, RPM11640 medium andEagle's MEM medium which are generally used or these culture mediacontaining a fetal bovine serum can be used.

[0169] The cultivation of the cells is conducted in the presence of 5%CO₂. The temperature for the cultivation is preferably 35 to 37° C., andthe period for the cultivation is usually 3 to 7 days.

[0170] L-Proline is suitably added to the media in such a manner thatits concentration may be from 5 to 1000 mM, preferably from 20 to 200mM, whereby the intended L-proline-3-hydroxylases can be produced moreefficiently.

[0171] Antibiotics such as kanamycin, penicillin or the like may beadded to the medium during the cultivation, if required.

[0172] A considerable amount of L-proline-3-hydroxylase is produced andaccumulated in the thus-cultivated transformants in comparison to themicroorganism strain used as the gene source, such as Streptomyces sp.TH1 or the like. Thus, the isolation and purification of the enzyme orthe production of cis-3-hydroxy-L-proline from L-proline using theenzyme can be performed far more efficiently in comparison to theproduction of cis-3-hydroxy-L-proline from L-proline using the nongenetically-engineered microorganism as the enzyme source, such asStreptomyces sp. TH1 or the like.

[0173] The production of L-proline-3-hydroxylase in the transformantscan be carried out by adding the culture, the cells or the treated cellsto an aqueous medium suitable for the enzymatic reaction together withL-proline, a divalent iron ion and 2-ketoglutaric acid, and adding asurfactant or an organic solvent, if required, to determinecis-3-hydroxy-L-proline produced. With respect to the activity of theL-proline-3-hydroxylase of which the formation is confirmed in the cell,the activity of the enzyme for producing 1 nmol ofcis-3-hydroxy-L-proline for 1 minute under the following conditions isdefined as 1 unit (U). The microorganism cells and the animal cells arehere called cells.

[0174] Measurement of L-proline-3-hydroxylase activity:

[0175] The cells, the treated cells or the enzyme preparation are addedto 240 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]buffer containing 12 mM L-proline, 24 mM 2-ketoglutaric acid, 4 mMferrous sulfate and 8 mM L-ascorbic acid to make 250 μl in total. Themixture is kept at 35° C. for 10 minutes. The reaction mixture is heatedat 100° C. for 2 minutes to stop the reaction, and the amount ofcis-3-hydroxy-L-proline produced in the reaction mixture is determinedby HPLC.

[0176] For the determination, any method capable of determining theamount of cis-3-hydroxy-L-proline may be employed. For instance,generally usable are (1) a post-column derivatization method and (2) apre-column derivatization method as mentioned above.

[0177] The enzyme may be isolated and purified in a usual manner fromthe culture of the transformant in which the formation ofL-proline-3-hydroxylase is confirmed in the cultivated cell as mentionedabove. For instance, the culture broth of the transformant iscentrifuged to collect the cultivated cells therefrom, and the cells arewashed and then disrupted by an ultrasonic cell disrupter, a Frenchpress, a Manton-Gauline homogenizer, a Dyno mill or the like to obtain acell-free extract. The purified enzyme preparation can be obtained byammonium sulfate precipitation, anion exchange chromatography such asdiethylaminoethyl (DEAE) Sepharose or the like, hydrophobicchromatography such as butyl-Sepharose, phenyl-Sepharose or the like,gel filtration, electrophoresis such as isoelectric pointelectrophoresis, and so on from the supernatant of the cell-free extractobtained by centrifugation.

[0178] The cultivated transformant cells that have been identified tocontain the L-proline-3-hydroxylase as formed therein can be cultivatedunder the same conditions as above, under which the transformant wascultivated, to thereby make the cells produce and accumulatecis-3-hydroxy-L-proline in the cells, and the thus-producedcis-3-hydroxy-L-proline can be collected from the culture to obtain it.

[0179] If the transformant cells derived from host cells which have theability of producing L-proline from saccharide sources and accumulatingit in the cultures and where such cells are used, it is possible toproduce cis-3-hydroxy-L-proline even if L-proline is not added to themedia during the cultivation of the cells therein. However, it isdesirable to suitably add to the media L-proline at a concentration offrom 5 to 1000 mM, preferably from 20 to 200 mM, whereby the intendedL-proline-3-hydroxylases can be produced more efficiently.

[0180] If the transformant cells have the ability of producing2-ketoglutaric acid from saccharide sources and accumulating it in thecultures and where such cells are used, it is possible to producecis-3-hydroxy-L-proline even if 2-ketoglutaric acid is not added to themedia during the cultivation of the cells therein. Where suchtransformant cells are used, saccharide sources such as glucose, etc.may be suitably added to the media to make the cells produce andaccumulate 2-ketoglutaric acid in the cultures, whereby the intendedL-proline-3-hydroxylases can be produced more efficiently. Where, on theother hand, transformant cells not having the ability of producing2-ketoglutaric acid from saccharide sources are used, 2-ketoglutaricacid may be added to the media during the cultivation of the cells, ifdesired.

[0181] If desired, 2-ketoglutaric acid and divalent iron ions may beadded to the media during the cultivation of the transformant cells.

[0182] To produce cis-3-hydroxy-L-proline, also employable is anothermethod to be mentioned below using, as the enzyme source, the culturesof the transformant cells where the formation ofL-proline-3-hydroxylases has been identified, the cells isolated fromthe cultures, or the products as obtained by processing the cells.

[0183] The method to produce cis-3-hydroxy-L-proline is as follows: Thecultures of the transformant cells, the cells isolated from the culture,or the products as obtained by processing the cells are added to aqueousmedia suitable for enzymatic reaction, along with L-proline, divalentiron ions and 2-ketoglutaric acid and optionally with surfactants andorganic solvents, thereby converting L-proline intocis-3-hydroxy-L-proline, and thereafter the resultingcis-3-hydroxy-L-proline is collected from the reaction mixtures.

[0184] As examples of the processed cells, dried cells, lyophilizedcells, surfactant-treated cells, enzymatically-treated cells,ultrasonically-treated cells, mechanically-ground cells,mechanically-compressed cells, solvent-treated cells, fractionated cellproteins, immobilized cells, immobilized materials obtained byprocessing their cells, etc. can be used. The enzymes preparationobtained by extraction from the cells having L-proline-3-hydroxylaseactivity, purified products of these enzymes, and immobilized productsthereof can also be used.

[0185] As examples of the aqueous medium, water, buffers such asphosphates, carbonates, acetates, borates, citrates and tris-buffers,alcohols such as methanol and ethanol, esters such as ethyl acetate,ketones such as acetone, and amides such as acetamide can be mentioned.

[0186] As examples of the surfactant, cationic surfactants such aspolyoxyethylene-stearylamine (for example, Nymeen S215 produced byNippon Oils & Fats Co.), cetyltrimethylammonium bromide, Cation FB,Cation F2-40E, etc.; anionic surfactants such as sodiumoleylamidosulfate, Newrex TAB, and Rapizole 80.; ampholytic surfactantssuch as polyoxyethylene-sorbitan monostearate (for example, NonionST221) or the like.; and also other tertiary amines PB,hexadecyldimethylamine, etc. can be mentioned. Any and every surfactantthat promotes the reaction may be employed. The concentration of thesurfactant is usually from 0.1 to 50 mg/liter, preferably from 1 to 20mg/liter.

[0187] As examples of the organic solvent, toluene, xylene, aliphaticalcohols, benzene and ethyl acetate can be mentioned. The concentrationof the organic solvent is usually from 0.1 to 50 μl/ml, preferably from1 to 20 μl/ml.

[0188] The reaction may be conducted during the cultivation of thetransformant having the activity of L-proline-3-hydroxylase, or may alsobe conducted after the completion of the cultivation, in the aqueousmedium using the cells, the treated cells, the purified enzyme or thecrude enzyme prepared from the culture.

[0189] The amount of the enzyme added to the reaction mixture isdetermined depending on the amount of the substrate used. It is usuallyfrom 1.0 to 10,000,000 U/liter, preferably from 1,000 to 3,000,000U/liter. In case of using the cells or the treated cells of themicroorganism, the concentration of wet cells is usually from 1 to 300g/liter.

[0190] The reaction is usually conducted at a temperature from 15 to 50°C. at a pH from 6.0 to 9.0 for 1 to 96 hours.

[0191] The concentration of L-proline used in the reaction may be from 1mM to 2 M. L-Proline can be supplied by adding L-proline itself to thereaction mixture, or adding the culture of the microorganism which canproduce and accumulate L-proline from sugar source. Further, if amicroorganism having the ability of producing L-proline from a sugarsource is used as the host microorganism of the transformant, L-prolineproduced from a sugar source by the host microorganism can be used inthe reaction. That is, L-proline produced by the transformant derivedfrom the host microorganism having the ability of producing L-proline isconverted into cis-3-hydroxy-L-proline in the culture usingL-proline-3-hydroxylase produced by the transformant, wherebycis-3-hydroxy-L-proline can be produced in the culture broth without theaddition of L-proline.

[0192] The divalent iron ion is required for the reaction. This divalentiron ion is ordinarily used in a concentration of from 1 to 100 mM. Anydivalent iron ion can be used so long it does not inhibit the reaction.As examples of the divalent iron ion, sulfates such as ferrous sulfate;chlorides such as ferrous chloride; ferrous carbide; and organic acidsalts such as citrates, lactates and fumarates can be mentioned. Whenthe divalent iron ion is contained in the cells, the treated cells orthe reaction mixture, the divalent iron is need not be added.

[0193] 2-Ketoglutaric acid itself may be added to the reaction mixtureor may be supplied from a compound which can be converted into2-ketaglutaric acid by the metabolic activity of the cells or thetreated cells used. As examples of such a compound, saccharides such asglucose; amino acids such as glutamic acid; and organic acids such assuccinic acid can be mentioned. These compounds may be used singly or incombination.

[0194] Cis-3-hydroxy-L-proline is recovered from the culture or theaqueous medium by any ordinary separation method, for example, columnchromatography using an ion-exchange resin, etc. by crystallization,etc.

[0195] The structure of the thus-recovered cis-3-hydroxy-L-proline canbe identified by ordinary analytical method such as ¹³C-NMR spectrum,¹H-NMR spectrum, mass spectrum, specific rotation or the like.

[0196] The cis-3-hydroxy-L-proline produced by the present invention canbe determined quantitatively by the above-mentioned post-columnderivatization method or pre-column derivatization method.

[0197] The present invention is illustrated more specifically byreferring to the following Examples.

EXAMPLE 1

[0198] (1) Production of Cis-3-hydroxy-L-proline:

[0199] SR3 medium comprising 1.0% glucose, 1.0% soluble starch, 0.5%yeast extract, 0.5% tryptone, 0.3% meat extract and 0.05% magnesiumphosphate, and adjusted to pH 7.2 with 6N NaOH, was put in test tubes(diameter 25 mm×length 200 mm) in an amount of 10 ml each and sterilizedat 120° C. for 20 minutes. One loopful of cells of Streptomyces sp. TH1,that had grown in HT-agar plate medium comprising 1% soluble starch,0.2% NZ amine, 0.1% yeast extract, 0.1% meat extract and 1.5% agar,adjusted to pH 7.2 with 6N NaOH, and sterilized at 120° C. for 20minutes, was inoculated into the above-mentioned SR3 medium in each testtube and cultivated at 28° C. for 2 days by shaking. The resultingculture was used as the 1st seed culture in the following step.

[0200] SR3 medium was put in 2 liter-Erlenmeyer flasks in an amount of200 ml each and sterilized at 120° C. for 20 minutes. Theabove-mentioned 1st seed culture was inoculated in the SR3 medium ineach Erlenmeyer flask and cultivated at 28° C. for 2 days by shaking.The resulting culture was used as the 2nd seed culture in the followingstep.

[0201] Df3 medium comprising 5% soluble starch, 3% corn steep liquor,0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate 7 hydrateand 0.5% calcium carbonate, and adjusted to pH 7.0 with 6N NaOH, was putin 5 liter-jar fermenters in an amount of 2 liters each and sterilizedat 120° C. for 20 minutes. The above-mentioned 2nd seed culture wasinoculated in the DF3 medium in each jar fermenter under germ-freecondition and cultivated under the condition of 700 rpm and 1 vvm, at28° C. for 2 days. During the cultivation, the pH of the medium was notadjusted. The thus-obtained culture was subjected to centrifugation at15,000×g for 10 minutes at 4° C. and 75 g of the wet cells thusseparated were obtained per liter of the culture. The wet cells werewashed with a physiological saline at 4° C. and then recentrifuged. Ifdesired, the thus-obtained wet cells were frozen and stored at −80° C.,and the fozen cells were thawed before use.

[0202] One gram of the thus-obtained wet cells was suspended in 10 ml ofa reaction mixture (a) [prepared by adding 1.4% (v/v) of Nymeen solution(prepared by dissolving 4 g of Nymeen S-215 (produced by Nippon Oils &Fats Co.) in 10 ml of xylene) to 100 mM TES buffer (pH 7.5) containing 5mM L-proline, 5 mM 2-ketoglutaric acid, 5 mM L-ascorbic acid and 1 mMferrous sulfate] and the mixture was allowed to stand at 30° C. for 5hours.

[0203] After the reaction, the cells were removed from the reactionmixture by centrifugation, and the amount of cis-3-hydroxy-L-prolineproduced in the supernatant was determined.

[0204] The determination was carried out by post-column derivatizationmethod with NBD by HPLC under the conditions mentioned below. Toidentify the intended product, the resulting product was eluted from thecolumn and reacted with NBD in the column line to form itsNBD-derivative and the derivative was determined by spectrofluorometry.

[0205] Conditions for determination by HPLC

[0206] [1] Apparatus used:

[0207] High Performance Liquid Chromatoigraphy Device (produced byShimadzu Seisakusho K.K.) Chromatopac CR6A System controller SCL-6BAutoinjector SIL-6B Liquid Chromatograph Pump LC-6A Column Oven CTO-6AChemical Reaction Box CRB-6A Spectrofluorometric Detector RF-550A

[0208] [2] Column Used:

[0209] SUMCHIRAL OA5000 (diameter 4.5 mm×length 250 mm, produced bySumika Chemical Analysis Service Limited)

[0210] [3] Conditions for Analysis: 1) Mobile Phase: aqueous solution of  1 mM copper sulfate 2) Flow Rate  1.0 ml/min of Mobile Phase: 3)Column 38° C. Temperature: 4) Buffer: 0.3 M boric acid buffer (pH 9.6) 25 mM ethylenediaminetetraacetic acid 5) Flow Rate  0.2 ml/min ofBuffer: 6) NBD Solution: methanol solution of  0.5 g/liter 7) Flow Rateof  0.5 ml/min NBD Solution: 8) Reaction 60° C. Temperature: 9) ReactionTime: about 3 min 10) Wavelength for excitation wavelength  503 nmDetection: emission wavelength  541 nm 11) Sample:   10 μl

[0211] As a result of the determination, it was verified that 910 μM(119 mg/liter) of cis-3-hydroxy-L-proline was produced in the reactionmixture.

[0212] (2) Production of Cis-3-hydroxy-L-proline:

[0213] Df3 medium was put into a test tube in an amount of 10 ml andsterilized at 120° C. for 20 minutes. One ml of the 1st seed cultureobtained in the same manner as in (1) of Example 1 was inoculated in theDf3 medium under germ-free conditions and cultivated at 28° C. for 2days by shaking. The resulting culture was centrifuged at 8000 rpm andat 4° C. for 10 minutes. The cells thus separated were suspended in 80mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer(pH 7.5), washed and then recentrifuged to separate the wet cells. Onegram of the thus-obtained wet cells was suspended in 10 ml of a reactionmixture (a) and the mixture was allowed to stand at 30° C. for 30minutes. After the reaction, the cells were removed from the reactionmixture by centrifugation, and the amount of cis-3-hydroxy-L-prolineformed in the supernatant was quantitatively determined.

[0214] As a result of the determination, it was verified that 1710 nM(224 μg/liter) of cis-3-hydroxy-L-proline was produced in the reactionmixture.

EXAMPLE 2

[0215] Purification of Cis-3-hydroxy-L-proline:

[0216] One hundred gram of the wet cells which were obtained in (1) ofExample 1 was suspended in 1 liter of the reaction mixture (a) asdescribed in (1) of Example 1, that had been put in a 2 liter-beaker.The suspension was allowed to stand at 30° C. for 5 hours with stirring.

[0217] After the reaction, the cells were removed from the reactionmixture by centrifugation, and cis-3-hydroxy-L-proline produced in thesupernatant were determined by the same method as described in (1) ofExample 1.

[0218] As a result of the determination, it was verified that 809 μM(106 mg/liter) of cis-3-hydroxy-L-proline was produced in the reactionmixture.

[0219] The supernatant separated from the reaction mixture was adjustedto pH 4.5, and passed through a column packed with 200 ml of anion-exchange resin, Diaion SK1B (NH⁴⁺ type, produced by Mitsubishi KaseiCo.). The eluted fractions containing cis-3-hydroxy-L-proline wasconcentrated under reduced pressure and then passed through a columnpacked with 20 ml of an ion-exchange resin, Diaion PA412 (OH-type,produced by Mitsubishi Kasei Co.). The eluted fraction containingcis-3-hydroxy-L-proline was concentrated under reduced pressure, andadjusted to pH 9.6, and 10 vol. % of o-phthalaldehyde (OPA) solution(0.075 g of OPA/ml ethanol solution) and 2 vol. % of β-mercaptoethanolsolution (10% v/v aqueous solution) were added thereto. The mixture wasthen allowed to stand at 60° C. for 5 minutes, whereby impurities ofprimary amino acids contained therein were reacted with OPA. Theresulting mixture was passed through a column packed with 10 ml ofSepabeads SP207 (produced by Mitsubishi Kasei Co.), to separatecis-3-hydroxy-L-proline from the impurities of OPA-derivatized primaryamino acids. The elued fraction containing cis-3-hydroxy-L-proline wasconcentrated under reduced pressure and again passed through a columnpacked with 20 ml of an ion-exchange resin, Diaion PA412 (OH-type,produced by Mitsubishi Kasei Co.) to separate a fraction containingcis-3-hydroxy-L-proline. The final fraction was concentrated and driedto obtain 68 mg of cis-3-hydroxy-L-proline as white crystals. The yieldof the product was 63%.

[0220] Physicochemical properties of the thus-obtained chemical compoundwere given below.

[0221] Specific rotation: [α]_(D) ²¹=−93.4 (c=0.503, H₂O)

[0222] FAB-mass spectrum: 132 (M+H)⁺

[0223]¹³C-NMR spectrum (D₂O, 125 MHz) ppm: 33.9, 44.5, 68.3, 71.6, 171.3

[0224]¹H-NMR spectrum (D₂O, 500 MHz) ppm: 2.18 (1H), 2.27 (1H), 3.52(1H), 3.62 (1H), 4.18 (1H), 4.77 (1H)

[0225] The above-mentioned molecular weight data of its mass spectrumand specific rotation, and the analytical results of its ¹³C-NMRspectrum and ¹H-NMR spectrum were found to be identical as the data asdescribed in the prior art [see J. Biol. Chem., 241, 1300 (1966), J.Antibiotics, 45, 824 (1992)] and the data of cis-3-hydroxy-L-prolinewhich was synthesized according to the methods of the prior art (seeLiebigs Ann. Chem., 1881 (1979), Tetrahedron, 42, 2421 (1986)].

EXAMPLE 3

[0226] Production of Cis-3-hydroxy-L-proline:

[0227] L-proline was hydroxylated by use of Streptomyces canus ATCC12647, Streptomyces canus ATCC 12646, Bacillus sp. TH2 and Bacillus sp.TH3.

[0228] SR3 medium was put in test tubes (diameter 25 mm×length 200 mm)in an amount of 10 ml each and sterilized at 120° C. for 20 minutes. oneloopful of cells of each of the microorganisms mentioned above that hadgrown in HT-agar plate medium was inoculated in the medium in a testtube and cultivated at 28° C. for 2 days by shaking. The resultingculture was used as a seed culture in the following steps.

[0229] Separately, Df4 medium comprising 2.5% glycerol, 2.5% glucose,1.5% soybean meal, 0.05% dipotassium hydrogen phosphate, 0.05% magnesiumsulfate 7 hydrate and 0.5% calcium carbonate, and adjusted to pH 7.0with 6N NaOH, was put in test tubes (diameter 25 mm×length 200 mm) in anamount of 10 ml each and sterilized at 120° C. for 20 minutes. One ml ofeach of the seed culture obtained above was inoculated in the medium ineach test tube under germ-free condition and cultivated at 28° C. for 1day by shaking. The thus-obtained cultures were subjected tocentrifugation at 15,000×g for 10 minutes at 4° C. The cells thusseparated were washed with 80 mM TES buffer (pH 7.5) and thenrecentrifuged respectively. The thus-obtained wet cells were suspendedin 1 ml of the reaction mixture (a) as described in (1) of Example 1respectively and allowed to stand at 30° C. for 3 hours.

[0230] As a result, it was verified that 242 μM, 202 μM, 318 μM and 141μM of cis-3-hydroxy-L-proline were produced in the reaction mixture byuse of the strains ATCC12647, ATCC12646, TH2 and TH3 respectively.

EXAMPLE 4

[0231] Production of Cis-3-hydroxy-L-proline:

[0232] In the same manner as in Example 3 except that Df2 mediumcomprising 5% soluble starch, 1.5% dry yeast, 0.05% potassium dihydrogenphosphate, 0.05% magnesium sulfate 7 hydrate and 0.5% calcium carbonateand adjusted to pH 7.0 with 6N NaOH, and further containing 0.1%L-proline was used in place of Df4 medium, each of Bacillus sp. TH2 andTH3 were cultivated.

[0233] As a result, it was verified that 745 μM (97.6 mg/l) ofcis-3-hydroxy-L-proline for the strain TH2 and 327 μM (42.8 mg/l) forstrain TH3 were produced in the aqueous medium.

EXAMPLE 5

[0234] Isolation and Purification of L-proline-3-hydroxylase:

[0235] (1) Preparation of Cell-free Extract:

[0236] Thirty grams of the wet cells obtained in (1) of Example 1 wassuspended in 200 ml of Buffer (A) [20 mM piperazine buffer, pH wasadjusted to 5.3 with 6N HCl, containing 1 mM dithiothreitol (DTT), 0.2mM EDTA, 0.1% (v/v) Tween 20 and 10% (v/v) glycerol] while cooling withice. The cells in the resulting suspension was disrupted by means of anultrasonic cell disrupter while cooling with ice. The thus-disruptedcell suspension was subjected to centrifugation at 30,000×g at 4° C. for30 minutes to separate a supernatant.

[0237] The subsequent operations were conducted under cooling with iceat a temperature of 4° C. or lower.

[0238] (2) Isolation and Purification by Column Chromatography:

[0239] (2)-1 Acid Treatment

[0240] The pH of the supernatant obtained in the previous step wasadjusted at 4.5 with 6 N HCl, and the precipitate formed was removedtherefrom through centrifugation as conducted at 15,000×g for 30 minutesto obtain a supernatant.

[0241] (2)-2 Resource Q Column Chromatography (I):

[0242] The supernatant obtained in the previous step was passed througha RESOURCE™ Q column (6 ml; produced by Pharmacia Co.) that had beenequilibrated with Buffer (A). The column was washed with Buffer (A), andthe fraction containing the desired enzyme was eluted with Buffer (A)having a linear concentration gradient of NaCl of 0 to 0.3M.

[0243] (2)-3 Resource Q Column Chromatography (II):

[0244] The active fraction obtained in the previous step was diluted3-fold with Buffer (B) [20 mM TES buffer (pH7.5) containing 1 mM DTT,0.2 mM EDTA and 10% (v/v) of glycerol] and was passed through aRESOURCE™ Q column (1 ml; produced by Pharmacia Co.) that had beenequilibrated with Buffer (B). After the column was washed with Buffer(B), the fraction containing the enzyme was eluted with Buffer (B)having a linear concentration gradient of NaCl of 0 to 0.3 M.

[0245] (2)-4 Phenyl Sepharose Column Chromatography:

[0246] NaCl was added to the active fraction obtained in the previousstep until an NaCl concentration was 2M. The mixture was applied to aPhenyl Sepharose column (1 ml; Phenyl Sepharose HP HiLoad) that had beenequilibrated with Buffer (B) containing 2M NaCl. The column was washedwith Buffer (B) containing 2M NaCl, and the fraction containing thedesired enzyme was eluted with Buffer (B) containing 0.1% (v/v) of Tween20.

[0247] (2)-5 Resource Q Column Chromatograph (III):

[0248] The active fraction obtained in the previous step was de-saltedusing a PD-10 column (1 ml; produced by Pharmacia Co.), and theresulting mixture was passed through a RESOURCE™ Q column (1 ml) thathad been equilibrated with Buffer (A). After the column was washed withBuffer (A), the fraction containing the desired enzyme was eluted withBuffer (A) having a linear concentration gradient of NaCl of 0 to 0.3 M.

[0249] (2)-6 Resource Q Column Chromatography (IV):

[0250] The active fraction obtained in the previous step was diluted3-fold with Buffer (B) containing 0.1% (v/v) Tween 20 and passed througha RESOURCE™ Q column (1 ml; produced by Pharmacia Co.) that had beenequilibrated with Buffer (B). The fraction containing the desired enzymewas eluted with Buffer B having a linear concentration gradient of NaClof 0 to 0.3M.

[0251] The foregoing steps for the isolation and purification of theL-proline-3-hydroxylase are summarized in Table 9. TABLE 9 Summary ofIsolation and Purification of L-proline-3-Hydroxylase Total TotalRelative Protein Activity Activity Yield Fraction (mg) (U) (U/mgprotein) (%) Cell-free 542 3540 6.5 100 Extract Acid-treatment 106 180017 56 Supernatant, pH 4.5 Resource Q(I), 7.5 1553 207 44 pH 5.3 ResourceQ(II), 3.3 1036 306 29 pH 7.5 Phenyl- 0.43 188 437 5.3 SepharoseResource 0.16 90.5 566 2.6 Q(III), pH 5.3 Resource Q(IV), 0.035 60.31723 1.7 pH 7.5

EXAMPLE 6

[0252] Properties of L-proline-3-Hydroxylase:

[0253] (1) Analysis by Electrophoresis:

[0254] The purified enzyme preparation obtained in Example 5 wasanalyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis,using polyacrylamide gel PAGEL NPU-12.5L produced by Atto Co. andSDS-PAGE Molecular Weight Standard, Broad Range produced by Biorad Co.As a result, it was verified that the enzyme was composed of almosthomogeneous sub-units having a molecular weight of approximately35,000±5,000 daltons.

[0255] (2) Properties of the Enzyme:

[0256] Using the reaction mixture mentioned below, the enzyme wassubjected to the substrate omission and addition tests, by which thecompounds indispensable to the enzyme reaction for hydroxylatingL-proline at the 3-position of L-proline, the promoters for the enzymereaction and the inhibitors against the enzyme reaction wereinvestigated.

[0257] The reaction mixture was composed of 100 mM TES buffer (pH 7.0),5 mM L-proline, 5 mM2-ketoglutaricacid, 1 mM ferrous sulfate, 5 mML-ascorbic acid and a predetermined amount of the pure enzyme, the totalvolume being 100 μl. The reaction was initiated by addition of theenzyme and continued for 10 minutes at 35° C. The reaction was stoppedby heating the reaction mixture at 100° C. for 2 minutes. The amount ofcis-3-hydroxy-L-proline formed in the reaction mixture was determined bythe pre-column derivatization method using HPLC. One hundred microlitersof 0.3 M boric acid buffer (pH 10.7), 4 μl of an aqueous solution of 10%(v/v) mercaptoethanol and 16 μl of ethanol solution of 5% (w/v) OPA wereadded to 100 μl of the reaction mixture, and the mixture was allowed tostand at 60° C. for 30 seconds. In addition, 50 μl of ethanol solutionof 2% (w/v) NBD was added thereto and the mixture was allowed to standat 60° C. for 40 minutes. The reaction was stopped by adding 30 μl of 1N HCl to the reaction mixture, and the precipitates formed were removedtherefrom by centrifugation and filtration. The cis-3-hydroxy-L-prolineformed by the reaction was quantitatively determined by HPLC analysis.

[0258] The HPLC was carried out for the determination under thefollowing conditions:

[0259] Mobile Phase: 10 mM Citric Acid (pH 4.0)/Methanol=3/1 (v/v)

[0260] Flow Rate: 1 ml/min

[0261] Column:YMC Pack ODS AQ-312 (produced by YMC Co., 6×150 mm)

[0262] Column Temperature: 50° C.

[0263] Detection: Fluorophotometry (excitation wavelength: 503 nm,emission wavelength: 541 nm)

[0264] The test results verified that L-proline, 2-ketoglutaric acid andFe⁺⁺ ion are indispensable for the enzyme reaction for hydroxylatingL-proline at the 3-position of L-proline, that L-ascorbic acid promotesthe enzyme reaction and that Zn⁺⁺, Cu⁺⁺, Co⁺⁺, Ba⁺⁺ and EDTA inhibit thereaction.

[0265] The test results are shown in Table 10. TABLE 10 Investigation ofComponents Influencing Enzyme Reaction of L-Proline-3-HydroxylaseComponents in Added (+)²⁾ Relative Reaction Mixture Not Added (−)Activity³⁾ Basic Reaction 100 Mixture¹⁾ −Pure Enzyme 0 −L-Proline 0−2-Ketoglutaric Acid 0 −Fe⁺⁺ 0 −L-Ascorbic Acid 25 +2 mM EDTA 5 +1 mmZn⁺⁺ 0 +1 mm Cu⁺⁺ 4 +1 mm Co⁺⁺ 13 +1 mm Ba⁺⁺ 42

[0266]1) The standard reaction mixture was composed of 100 mM TES buffer(pH 7.0), 5 mM L-proline, 5 mM 2-ketoglutaric acid, 1 mM ferroussulfate, 5 mM L-ascorbic acid and a pre-determined amount of the pureenzyme, the total volume being 100 μl. The reaction was carried out at35° C. for 10 minutes.

[0267] 2) “(+)” means that the reaction mixture contained the componentshown in the table. “(−)” means that the reaction mixture did notcontain the component shown in the table. The concentration shown in thetable means the concentration of the component in the reaction mixture.

[0268] 3) The activity is indicated as the relative activity to theactivity in the standard reaction mixture being defined as 100.

[0269] (3) Optimum pH Range:

[0270] In the above-mentioned method of determining the enzymaticactivity of the L-proline-3-hydroxylase, the reaction was carried outwhile the buffer component in the reaction mixture was changed to MESbuffer [2-(N-morpholino)ethanesulfonic acid] at pH of 5.5 to 6.5, it waschanged to PIPES buffer [piperazine-N,N′-bis(2-ethanesulfonic acid)] atpH of 6.5 to 7.5, it was changed to TES buffer at pH of 7.0 to 8.0, andit was changed to TAPS buffer(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid] at pH of 8.0to 9.0. As a result, the enzyme had an activity of more than 80% of themaximum activity thereof at pH ranging from pH 6.5 to 7.5. The detailedtest results are shown in Table 11 below. TABLE 11 Optimum pH Range forthe Enzyme Reaction pH (buffer) Relative Activity¹⁾ 5.5 (MES) 15.0 6.0(MES) 70.9 6.5 (PIPES) 83.3 7.0 (PIPES) 92.1 7.0 (TES) 100.0 7.5 (PIPES)80.4 7.5 (TES) 85.0 8.0 (TES) 72.2 8.0 (TAPS) 76.3 8.5 (TAPS) 52.9 9.0(TAPS) 44.5

[0271] (4) Stable pH Range:

[0272] The enzyme solution in Buffer (B) was diluted 3-fold with 100 mMof a buffer (MES buffer at pH of 5.5 to 6.5, PIPES buffer at pH of 6.1to 7.5, TES buffer at pH of 7.0 to 8.0, TAPS buffer at pH of 8.0 to9.0), kept at 4° C. for 23 hours, and then its activity was determined.The enzyme kept at pH ranging from 6.5 to 8.0 had an activity of 80% ormore of the original activity of the enzyme before the test.Accordingly, the enzyme was kept stable at pH ranging from 6.5 to 8.0.

[0273] (5) Optimum Temperature Range:

[0274] In the above-mentioned of determining the enzyme activity of theL-proline-3-hydroxylase, the reaction was carried out at a temperatureranging from 15 to 50° C. for 15 minutes. As a result, the enzyme had anactivity of 80% or more of the maximum activity thereof at temperaturesranging from 35 to 40° C. The detailed test results are shown in Table12 below. TABLE 12 Optimum Temperature Range for the Enzyme ReactionReaction Relative Temperature (° C.) Activity¹⁾ 15 19 20 29 25 53 30 7435 100 40 89 45 64 50 28

[0275] (6) Stable Temperature Range:

[0276] The enzyme was kept in Buffer (B) at temperatures ranging from 0to 60° C. for 30 minutes and thereafter the activity of the enzyme wasdetermined. As a result, the enzyme was inactivated at 50° C. or higherfor 30 minutes.

[0277] (7) Km Value;

[0278] The Km value is 0.49 mM for L-proline and is 0.11 mM for2-ketoglutaric acid, when determined in a TES buffer (pH 7.0) containing5 mM L-ascorbic acid, 1 mM ferrous sulfate and a pre-determined amountof this enzyme.

[0279] (8) Isoelectric Point:

[0280] The enzyme was analyzed, using Phast system (produced byPharmacia Co.), to determine the isoelectric point of the enzyme. As aresult, the isoelectric point of the enzyme was 4.3.

[0281] (9) N-terminal Amino Acid Sequence:

[0282] The enzyme was analyzed, using Protein Sequencer Model PPSQ-10(produced by Shimadzu Seisakusho K.K.), to determine the N-terminalamino acid sequence of the enzyme. The result is as follows: SequenceNo. 5: (N-terminal) 1 MetArgSerHisIleLeuGlyArgIleGlu 11LeuAspGlnGluArgLeuGlyArgAspLeu 21 GluTyrLeuAlaThrValProThrVal

EXAMPLE 7

[0283] Production of Cis-3-hydroxy-L-proline:

[0284] The enzyme reaction by use of purified L-proline-3-hydroxylaseobtained in Example 5 was carried out. The reaction mixture was composedof 200 mM TES buffer (pH 7.0), 20 mM L-proline, 20 mM 2-ketoglutarate, 5mM L-ascorbic acids 1 mM, ferrous sulfate, and 106 U of purified enzymepreparation, the total volume being 100 μl. The reaction was carried outat 35° C. for 30 minutes. As a result of the reaction, 18 mM (2.4 g/l)of cis-3-hydroxy-L-proline was produced in the reaction mixture.

EXAMPLE 8

[0285] Preparation of partial DNA fragment of the gene encodingL-proline-3-hydroxylase protein derived from Streptomyces sp. TH1:

[0286] (1) Isolation of Chromosomal DNA of Streptomyces sp. TH1:

[0287] Chromosomal DNA of Streptomyces sp. TH1 was isolated in the usualmanner as follows. SK#2 medium (comprising 0.25% glucose, 1.0% solublestarch, 0.25% yeast extract, 0.25% peptone, 0.15% meat extract, 0.01%potassium dihydrogen phosphate and 0.03% magnesium sulfate, and adjustedto a pH 7.6 with 6 N NaOH) containing 5% mannitol and 0.05% glycine, wasput in test tubes in an amount of 10 ml each, and sterilized at 120° C.for 20 minutes. One loopful of cells of Streptomyces sp. TH1 which hadgrown in TH-agar plate medium was inoculated in the above-mentioned SK#2medium, and cultivated at 28° C. for 3 days with shaking.

[0288] The culture was centrifuged, and the obtained cells were washedwith 10 ml of a 10.3% sucrose solution, and suspended in 6 ml of TScomprising 10.3% sucrose, 50 mM tris-HCl (pH 8.0) and 25 mM EDTA. Onemilliliter of a lysozyme solution (50 mg/ml-TS) was added thereto, andthe mixture was incubated at 37° C. for 60 minutes. Subsequently, 0.6 mlof a Proteinase K (produced by Sigma Co.) solution (2 mg/ml-TS) wasadded to the lysozyme-treated solution, and gently stirred. Further, 3.6ml of a 3.3% (w/v) SDS solution was added thereto while gently mixing,and the mixture was incubated at 37° C. for 60 minutes. The mixture washeated at 50° C. for 30 minutes, and then cooled with water. An equalamount of TE [containing 10 mM tris-HCl (pH 8.0) and 1 mM EDTA]saturated phenol-chloroform (1/1, v/v) was added thereto, and the mixedsolution was moderately shaked for 30 minutes. After the centrifugation,the upper layer was taken, and again subjected to extraction with themixture of TE saturated phenol-chloroform. The extract was centrifuged,and an equal amount of chloroform was then added to the upper layer, andmixed. The mixture was recentrifuged. The upper layer was taken, and 20μl of an RNase A aqueous solution (10 mg/ml) heat-treated at 100° C. for10 minutes were added to the upper layer. The mixture was incubated at37° C. for 45 minutes. To the RNase A-treated solution were added 1/10volume of a 5 M NaCl aqueous solution and 1/4 volume of 50% PEG6000, andgently mixed. The mixture was allowed to stand overnight while beingcooled with ice. After the mixed solution was centrifuged at 12,000 rpmfor 10 minutes, the supernatant was discarded completely, and theprecipitate was dissolved in 5 ml of TE. After 1/10 volume of a 3 Msodium acetate solution and 1/30 volume of a 66 mM magnesium chloridesolution were added thereto and mixed, 2.2 volumes of cold ethanol wereadded, and gently mixed. After the mixed solution was centrifuged at10,000 rpm for 10 minutes, the supernatant was discarded, and theprecipitate was washed twice with 70% cold ethanol. The precipitatecontaining 250 μg of chromosomal DNA was dissolved in TE and used in thesubsequent experiment as chromosomal DNA.

[0289] (2) Determination of Partial Amino Acid Sequence of Streptomycessp. TH1-derived L-proline-3-hydroxylase Protein:

[0290] The N-terminal amino acid sequence of the purified enzymeobtained in Example 5 was sequenced, using Protein Sequencer ModelPPSQ-10 (produced by Shimadzu Seisakusho Co.), to determine theN-terminal amino acid sequence thereof as indicated by Sequence No. 5.

[0291] Furthermore, the purifed enzyme obtained in Example 5 was treatedwith lysyl endopeptidase in 0.1 M Tris-HCl with 4 M urea (pH 9), at 37°C. for 6 hours, and the thus-obtained peptide fragments were collectedthrough HPLC. Their amino acid sequences were also analyzed in the samemanner as above to determine the partial amino acid sequences asindicated by Sequence Nos. 6 and 7.

[0292] (3) Preparation of Partial DNA of L-proline-3-hydroxylase Gene:

[0293] A sense chain mix DNA primer as indicated by Sequence No. 8,which corresponds to the sequence of from the 1st to 6th amino acids inthe amino acid sequence as indicated by Sequence No. 5, and ananti-sense chain mix DNA primer as indicated by Sequence No. 9, whichcorresponds to the sequence of from the 24th to 28th amino acids in theamino acid sequence as indicated by Sequence No. 5, were synthesized,using 380A DNA Synthesizer (produced by Applied Biosystems Co.).

[0294] Using the above-synthesized DNAs as primers and the Streptomycessp. TH1 chromosome DNA as template, the PCR was conducted using DNAThermal Cycler 480 (produced by Perkin Elmer Cetus Co.).

[0295] The reaction was conducted using 20 μl of a reaction mixturehaving the following formulation.

[0296] Formulation of the reaction mixture:

[0297] 21 ng/μl of Streptomyces sp. TH1 chromosome DNA,

[0298] 10 μM of sense chain mix DNA primer,

[0299] 10 μM of anti-sense chain mix DNA primer,

[0300] 0.125 U/μl of Pfu DNA polymerase

[0301] (produced by STRATAGENE Co.),

[0302] 10% of DMSO,

[0303] 20 mM of Tris-HCl (pH 8.2),

[0304] 10 mM of KCl,

[0305] 6 mM of ammonium sulfate,

[0306] 2 mM of magnesium chloride,

[0307] 0.1% of Triton X-100, and

[0308] 10 ng/μl of bovine serum albumin.

[0309] After the completion of an incubation at 95° C. for 5 minutes, athree step incubation, namely at 95° C. for 0.5 minute, at 25° C. for0.5 minute and at 75° C. for 1 minute was repeated for a total of 40times. The reaction mixture was subjected to electrophoresis with 15%polyacrylamide gel (PAGEL NPU-15L produced by Atto Co.), and theamplified 83 bp DNA was recovered using da Vinci Kun (Pen Touch RecoveryNB-7000 Model) manufactured by Nippon Eido K. K.

[0310] Using the recovered DNA fragment (83 bp) as a template, the sensestrand mix DNA primer indicated in Sequence No. 8 as a primer and theanti-sense strand mix DNA primer indicated in Sequence No. 10corresponding to amino acids Nos. 21st to 25th of an amino acid sequenceindicated in Sequence No. 5 as a primer, the PCR was conducted again inthe same manner, and the amplified 74 bp DNA was recovered. Thethus-recovered DNA fragment was inserted into a Sma I site of pUC18using Sure Clone Ligation Kit (produced by Pharmacia Co.), and thenucleotide sequence was determined by a nucleotide sequencing kit (TaqDyeDeoxy™ Terminator Cycle Sequencing Kit produced by AppliedBiosystems).

[0311] The nucleotide sequences thus determined are indicated bySequence Nos. 11 and 12. Thus, the two nucleotide sequences weredetermined. The amino acid sequence to be presumed from the nucleotidesequence of Sequence No. 11 completely corresponded to the N-terminalamino acid sequence of the pure enzyme as indicated by Sequence No. 1,except for the 2nd amino acid that could not be identified in thesequencing of the N-terminal amino acid sequence. The amino acidsequence to be presumed from the nucleotide sequence of Sequence No. 12exhibited high homology to the N-terminal amino acid sequence of thepure enzyme as indicated by Sequence No. 5.

EXAMPLE 9

[0312] Cloning of a DNA fragment containing L-proline-3-hydroxylasegene:

[0313] (1) Preparation of a Digoxigeniated (DIGated) Probe:

[0314] The two 74 bp fragments obtained in (3) of Example 8 andindicated by Sequence Nos. 11 and 12 each were inserted into the Sma Isite of pUC18. Using each of these two plasmids thus obtained as atemplate, each plasmid was subjected to PCR in the same manner as in (3)of Example 8, using 50 μl of a reaction mixture containing 10 μM of thesense chain synthetic DNA as indicated by Sequence No. 8 and 10 μM ofthe anti-sense chain synthetic DNA as indicated by Sequence No. 10. Theresulting reaction mixture each were subjected to 12.5% polyacrylamidegel electrophoresis to identify the formation of an amplified 74 bpfragment. From each gel, the amplified fragment was recovered in thesame manner as in (3) of Example 8.

[0315] The 74 bp fragment was digoxigeniated (DIGated), using PCR DIGLabeling Kit (produced by Boehringer Mannheim Co.).

[0316] Using each of the thus-recovered two 74 bp fragments as atemplate, each fragment was subjected to PCR, using 2.5 U of Pfu DNApolymerase (produced by STRATAGENE Co.), 5 μl of 510 buffer for Pfu DNApolymerase (produced by STRATAGENE Co.), 5 μl of DMSO, 5 μl of 510 PCRDIG mix (produced by Boehringer Mannheim Co.) and 50 μl of a reactionmixture comprising 10 μM of the sense chain synthetic DNA of SequenceNo. 8 and 10 μM of the anti-sense chain synthetic DNA of Sequence No.10.

[0317] After the incubation at 95° C. for 5 minutes, the step of theincubation at 95° C. for 0.5 minute, at 50° C. for 0.5 minute and at 75°C. for 1 minute was repeated 40 times. After the reaction, each reactionmixture was subjected to 12.5% polyacrylamide gel electrophoresis toidentify the formation of an amplified 74 bp fragment. From each gel,was recovered the amplified fragment in the same manner as in (3) ofExample 8. The resulting two amplified fragments were used as probes.The probe derived from the nucleotide sequence of Sequence No. 11 isreferred to as probe A, and that from the nucleotide sequence ofSequence No. 12 as probe B.

[0318] (2) Southern Hybridization:

[0319] Twenty units of a restriction enzyme Pst I (produced by TakaraShuzo Co., LTD) was added to 20 μg of Streptomyces sp. TH1 chromosomeDNA and reacted at 37° C. for 2 hours to cleave the DNA, which was thensubjected to agarose gel electrophoresis. Using the probe A as obtainedin (1) of Example 9, Southern hybridization was conducted with DIGLuminescent Detection Kit (produced by Boehringer Mannheim Co.)according to the method as described in the description attached to thekit.

[0320] Precisely, after the completion of the agarose gelelectrophoresis, the gel was gently shaken in 0.25 N HCl for 20 minutesand then blotted onto Hybond-N⁺ membrane (produced by Amersham Co.) in0.4 M sodium hydroxide solution, while being sucked at 7.5 mmHg usingGenopirator Pump AE-6680P (produced by ATTO Co.) and GenopiratorAE-6680C (produced by ATTO Co.). The thus-blotted membrane was dried inair. The resulting membrane was dipped in 10 ml of a hybridizationbuffer of DIG Luminescent Detection Kit [comprising 50% v/v offormamide, 2% of a blocking reagent, 0.1% w/v of N-laurylsarcosine and0.02% w/v of SDS in x5 SSC (×1 SSC is comprised of 150 mM sodiumchloride and 15 mM sodium citrate)] at 42° C. for 1 hour and thereafterdipped in a probe solution [prepared by adding 3 μl of the probe A asobtained in (1) of Example 9 to 200 μl of the hybridization buffer, thenheating the mixture at 95° C. for 2 hours and thereafter adding theretothe hybridization buffer until the total volume was 1.5 ml], at 42° C.overnight. The thus-obtained membrane was washed twice each with 25 mlof ×2 SSC containing 0.1% of SDS for 5 minutes at room temperature andthen twice each with 25 ml of ×0.1 SSC containing 0.1% of SDS for 15minutes at 68° C.

[0321] After this, the membrane was treated with a washing buffer[buffer 1 (0.1 M maleic acid, 0.15 M sodium chloride, pH 7.5) containing0.3% w/v of Tween-20] for 1 to 5 minutes at room temperature, then with50 ml of buffer 2 (buffer 1 containing 1% of a blocking reagent) for 30minutes at room temperature, then with 10 ml of buffer 2 containing 1 μlof anti-digoxigenin-AP Fab for 30 minutes at room temperature, thentwice each with 50 ml of buffer 2 for 30 minutes at room temperature,then with 10 ml of buffer 3 (0.1 M Tris-HCl, 0.1 M sodium chloride, 50mM magnesium chloride, pH 9.5) for from 2 to 5 minutes at roomtemperature, and then with 5 ml of buffer 3 containing 50 μl of LumigenPPD for 5 minutes at room temperature, in that order. Subsequently,water was removed from the film quickly over a filter paper, then thefilm was wrapped with a wrapping film, Saran Wrap (poly vinylidenechloride film) and thereafter allowed to stand at 37° C. for 15 minutes.Using Hyperfilm ECL (produced by Amersham Co.), the resulting film wasexposed at room temperature for 30 minutes.

[0322] A DNA fragment as strongly hybridized with the probe was detectedat the position of approximately 6 kb.

[0323] The same process as in (2) of Example 9 was repeated, while usingthe probe B as obtained in (1) of Example 9 and while using 20 U of arestriction enzyme KpnI (produced by Takara Shuzo Co., LTD) in place ofthe restriction enzyme PstI. As a result, a DNA fragment as stronglyhybridized with the probe was detected at the position of approximately5 kb.

[0324] (3) Fractionation of Chromosome DNA:

[0325] Twenty units of a restriction enzyme PstI (produced by TakaraShuzo Co., LTD) was added to 20 μg of Streptomyces sp. TH1 chromosomeDNA and the mixture was allowed to stand at 37° C. for 2 hours to cleavethe DNA, which was then mixed with the same amount, as that of thereaction mixture, of TE-saturated phenol/chloroform. The resultingmixture was centrifuged, and the upper layer was taken out. To the layerwas added 2.2 times volume of cold ethanol and then gently mixed. Thismixture was centrifuged at 10,000 rpm for 10 minutes, the supernatantwas removed, and the precipitate was washed twice with 70% cold ethanolto obtain an ethanol precipitate. The process of obtaining the ethanolprecipitate by using TE-saturated phenol/chloroform and cold ethanol ishereinafter referred to as an ethanol precipitation. The ethanolprecipitate was dissolved in 120 μl of TE and subjected to agarose gelelectrophoresis. After the completion of the electrophoresis, a DNAfraction at approximately 6 kb was extracted from the agarose gel andpurified by the use of Prep-A-Gene (produced by Biorad Co.). Thusapproximately 0.5 μg of a PstI-cleaved chromosome DNA fraction wasobtained.

[0326] The same process as in (3) of Example 9 was repeated, exceptusing 20 U of a restriction enzyme KpnI in place of PstI. Thus wasobtained approximately 0.5 μg of a KpnI-cleaved chromosome DNA fractionat approximately 5 kb.

[0327] (4) Formation of Plasmid Library:

[0328] 1) After 1 μg of pBluescriptII KS(+) (produced by STRATAGENE Co.)was cleaved with restriction enzyme PstI (produced by Takara Shuzo Co.,LTD) and then dephosphorylated with an alkaline phosphatase (derivedfrom calf intestines) (produced by Takara Shuzo Co., LTD) in accordancewith the method described in the description attached to the product.After the completion of the reaction, the reaction mixture was subjectedto the ethanol precipitation to obtain an ethanol precipitate. Theprecipitate was dissolved in 5 μl of TE. The thus-obtained, PstI-cleavedpBluescriptII KS(+) DNA was reacted with 0.1 μg of PstI-cleavedchromosome DNA as obtained in (3) of Example 9, using a ligation kit(TAKARA Ligation Kit, produced by Takara Shuzo Co., LTD), for 2.5 hoursat 26° C., whereby the two DNAs were ligated to each other. Using thethus-ligated DNA, E. coli XL2-Blue was transformed in a usual manner.The resulting transformant were spread on LB-agar medium containing 50μg/ml of ampicillin and cultivated thereon overnight at 37° C. to obtainabout 240 colonies.

[0329] 2) After 1 μg of pBluescriptII KS(+) (produced by STRATAGENE Co.)was cleaved with a restriction enzyme KpnI (produced by Takara ShuzoCo., LTD), dephosphorylation was conducted with an alkaline phosphatase(derived from calf intestines) (produced by Takara Shuzo Co., LTD) inaccordance with the method described in the description attached to theproduct. After the completion of the reaction, the reaction mixture wassubjected to the ethanol precipitation to obtain an ethanol precipitate.The precipitate was dissolved in 5 μl of TE. The thus-obtained,KpnI-cleaved pBluescriptII KS(+) DNA was reacted with 0.1 μg ofKpnI-cleaved chromosome DNA as obtained in (3) of Example 9, using aligation kit (TAKARA Ligation Kit, produced by Takara Shuzo Co., LTD),for 2.5 hours at 26° C., whereby the two DNAs were ligated to eachother. Using the thus-ligated DNA, E. coli XL2-Blue was transformed in ausual manner. The resulting transformant were spread on LB-agar mediumcontaining 50 μg/ml of ampicillin and cultivated thereon overnight at37° C. to obtain about 200 colonies.

[0330] (5) Selection of Intended Clone:

[0331] The colonies having the desired clone was selected from theabove-obtained colonies as follows.

[0332] Precisely, the colonies appearing on the LB-agar medium weretransferred onto a nylon membrane (Nytran, produced by Schleicher &Schuell Co.) that has been previously washed with ×5 SSC, and themembrane was put on filter paper immersed with 0.5 M NaOH for 5 minutes.Subsequently, the film was put on filter paper immersed with 1.0 MTris-HCl (pH 8.0) for 5 minutes and then on filter paper immersed with1.5 M sodium chloride-1.0 M Tris-HCl (pH 8.0) for 5 minutes. After thefilm was lightly washed with ×2 SSC, then crosslinking was conducted byexposure to ultraviolet rays (120 mJ/cm²). Then the surface of the filmwas wiped with ×2 SSC containing 0.1% of SDS, and the membrane was driedin air.

[0333] Using the DIGated probes as obtained in (1) of Example 9,positive colonies were detected on the membrane in accordance with theprocess as described in (2) of Example 9 using DIG Luminescent DetectionKit (produced by Boehringer Mannheim Co.).

[0334] Precisely, by the colony hybridization between about 240 coloniesas obtained in 1) of Example 9(4) and the probe A as obtained in (1) ofExample 9, one positive colony having the intended clone was detected.By the colony hybridization between about 200 colonies as obtained in 2)of Example 9(4) and the probe B as obtained in (1) of Example 9, fivepositive colonies each having the intended clone were detected.

[0335] From these colonies, plasmids were extracted in a usual manner.Thus were obtained pTH30, pTH71 and pTH75.

[0336] The structures of the plasmids were identified through digestionwith restriction enzymes. The structure of pTH30 was such that aPstI-cleaved DNA fragment of approximately 6 kb was inserted into thePstI site of pBluescriptII KS(+) (see FIG. 1); while the structures ofpTH71 and pTH75 were such that a KpnI-cleaved DNA fragment ofapproximately 5 kb was inserted into the KpnI site of pBluescriptIIKS(+) in the opposite directions (see FIG. 2).

[0337] (6) Determination of Base Sequence:

[0338] 1) Using a deletion kit for kilosequences (produced by TakaraShuzo Co., LTD), plasmid pTH30 was processed to give various deletionmutant plasmids. The reagents and the process as indicated in thedescription attached to the kit were employed. The nucleotide sequencesof the deletion plasmids were analyzed, using a base sequencing kit (TaqDyeDeoxy™ Terminator Cycle Sequencing Kit, produced by AppliedBiosystems Co.), and the nucleotide sequence of 1081 b KpnI-EcoRIfragment as indicated by Sequence No. 13 was identified.

[0339] In the nucleotide sequence thus identified, the nucleotidesequence indicated by Sequence No. 3 that codes for the protein composedof 290 amino acid residues indicated by Sequence No. 1 was present. Theamino acid sequence comprises the N-terminal amino acid sequenceindicated by Sequence No. 5, which corresponded to the sequence of thepure L-proline-3-hydroxylase, and the internal amino acid sequencesindicated by Sequence Nos. 6 and 7, which revealed the existence of theintended L-proline-3-hydroxylase gene in the PstI fragment ofapproximately 6 kb obtained hereinabove. The gene is hereinafterreferred to as L-proline-3-hydroxylase I-type gene.

[0340] 2) The nucleotide sequence of the Streptomyces-derived insertionfragment terminal in each of plasmids pTH71 and pTH75 was analyzed,using a base sequencing kit (Taq DyeDeoxy™ Terminator Cycle SequencingKit, produced by Applied Biosystems Co.). On the basis of thethus-sequenced nucleotide sequence, a sequencing primer was synthesizedusing 380A Model DNA Synthesizer (produced by Applied Biosystems Co.).Using the primer, the nucleotide sequence of a part of each of pTH71 andpTH75 existing downstream from the Streptomyces-derived insertionfragment was analyzed. On the basis of the thus-sequenced nucleotidesequence, another sequencing primer was synthesized. Using the primer,apart of each of the plasmids existing further downstream was analyzed.The base sequencing operation comprising the above-mentioned steps wasrepeated, by which the NruI-KpnI fragment of 1826 bp was sequenced as inSequence No. 14.

[0341] The nucleotide sequence of the fragment thus sequenced comprisedthe nucleotide sequence indicated by Sequence No. 4 that codes for theprotein composed of 290 amino acid residues as indicated by Sequence No.2. The nucleotide sequence of Sequence No. 4 comprised the nucleotidesequence indicated by Sequence No. 12. The nucleotide sequence indicatedby Sequence No. 4 was homologous at 78.5% to the L-proline-3-hydroxylaseI-type gene. The gene indicated by Sequence No. 4 is hereinafterreferred to as L-proline-3-hydroxylase II-type gene.

EXAMPLE 10

[0342] Construction of L-proline-3-hydroxylase Expression Plasmid:

[0343] (1) Construction of L-proline-3-hydroxylase I-type GeneExpression Plasmid:

[0344] Plasmid pTH30 as obtained in Example 9 was cleaved with arestriction enzyme EcoRI, and then self-ligated using a ligation kit(produced by Takara Shuzo Co., LTD). E. coli XL1-Blue MRF′ strain wastransformed with the DNA resulting from the self-ligation in a usualmanner. A plasmid was extracted from the transformant in a usual manner,and the structure of the plasmid was identified through digestion withrestriction enzyme.

[0345] As a result of the above, obtained was plasmid pTH40, from whichhad been removed the EcoRI fragment of approximately 3 kb existing inthe 3′-side of L-proline-3-hydroxylase gene (see FIG. 4).

[0346] Subsequently, plasmid pTH40 was cleaved with KpnI and SmaI, thena fragment of approximately 0.95 kbp containing L-proline-3-hydroxylaseI-type gene was identified through agarose gel electrophoresis, and thefragment was recovered from the agarose gel in a usual manner. TheKpnI-SmaI-cleaved fragment was inserted into the KpnI-SmaI-cleaved siteof pBluescriptII KS(+), using a ligation kit (produced by Takara ShuzoCo., LTD). With the resulting DNA, E. coli XL1-Blue MRF′ strain wastransformed in a usual manner. The resulting transformant was spread onLB-agar medium containing 50 μg/ml of ampicillin and cultivated thereonovernight at 37° C. A plasmid was extracted from the colonies of thetransformant thus grown in a usual manner, and its structure wasidentified through digestion with restriction enzyme.

[0347] As a result of the above, obtained was plasmid pTH50, into whichhad been inserted the DNA fragment coding for L-proline-3-hydroxylaseI-type gene in the same direction as that for transcription of lacpromoter (see FIG. 3).

[0348] (2) Construction of L-proline-3-hydroxylase II-type GeneExpression Plasmid:

[0349] Plasmid pTH75 as obtained in Example 9 was cleaved withrestriction enzymes NruI and SacI, and thus-cleaved fragment ofapproximately 1900 bp containing L-proline-3-hydroxylase II-type genewas recovered through agarose gel electrophoresis. The NruI-SacI-cleavedfragment was inserted into the HincII-SacI-cleaved site of pBluescriptIIKS(+), using a ligation kit (produced by Takara Shuzo Co., LTD). Withthe resulting DNA, E. coli XL2-Blue MRF′ strain was transformed in ausual manner. The resulting transformant was spread on LB-agar mediumcontaining 50 μg/ml of ampicillin and cultivated thereon overnight at37° C. A plasmid was extracted from the colonies of the transformantthus grown in a usual manner, and its structure was identified throughdigestion with restriction enzyme.

[0350] As a result of the above, obtained was plasmid pEX1-5, into whichhad been inserted the DNA fragment coding for L-proline-3-hydroxylaseII-type gene in the direction same as that for transcription of lacpromoter (see FIG. 4).

EXAMPLE 11

[0351] Production of L-proline-3-hydroxylase by Transformants:

[0352] (1) Production of L-proline-3-hydroxylase by Transformant HavingL-proline-3-hydroxylase I-type Gene:

[0353]E. coli ATCC12435 was transformed with plasmid pTH50 as obtainedin Example 10. The resulting transformant was inoculated in 3 ml of anLB medium containing 50 μg/ml of ampicillin and cultivated thereinovernight at 30° C. by shaking. The resulting culture was centrifuged.If desired, the thus-obtained wet cells were frozen and stored at −20°C., and the fozen cells were thawed before use.

[0354] Added to 250 μl of a reaction mixture [containing 12 mML-proline, 24 mM 2-ketoglutaric acid, 4 mM ferrous sulfate and 8 mML-ascorbic acid in 200 mM TES buffer (pH 7.5)] at 4% (w/v) in terms ofthe wet cells, and the mixture was allowed to stand for 10 minutes at35° C. The reaction mixture was heated at 100° C. for 2 minutes to stopthe reaction.

[0355] The resulting reaction mixture was centrifuged, and 100 μl of 0.3M borate buffer (pH 10.7), 4 μl of 10% (v/v) mercaptoethanol aqueoussolution and 16 μl of 5% (w/v) OPA in ethanol were added to 100 μl ofthe resulting supernatant and left at 60° C. for 30 seconds, and then 50μl of 2% (w/v) NBD in ethanol was added thereto. The mixture was allowedto stand at 60° C. for 40 minutes. Then 30 μl of 1 N hydrochloric acidwas added to the reaction mixture to stop the reaction. The resultingreaction mixture was centrifuged and filtered through a filter to removethe precipitate therefrom, and the resulting filtrate was analyzedthrough HPLC by which the product cis-3-hydroxy-L-proline produced wasquantitatively determined.

[0356] HPLC was conducted under the following conditions.

[0357] Mobile Phase: 10 mM citric acid (pH 4.0)/methanol=3/1 (v/v).

[0358] Flow Rate: 1 ml/min.

[0359] Column: YMC Pack ODS AQ-312 (produced by YMC Co., 6×150 mm).

[0360] Column Temperature: 50° C.

[0361] Detection: fluorophotometry

[0362] excitation wavelength: 503 nm

[0363] emission wavelength: 541 nm.

[0364] As is shown in Table 13 below, the transformant E. coliATCC12435/pTH50 produced L-proline-3-hydroxylase more by approximately34 times/cell than the Streptomyces sp. TH1 strain which had been usedas the gene source. TABLE 13 L-proline-3-hydroxylase Activity Producedby Transformant Cell Relative Strain Activity¹⁾ Activity²⁾ E. coliATCC12435/pTH50 1.94 34.0 E. coli ATCC12435/pBluescriptII Not — KS(+)detected. Streptomyces sp. TH1³⁾ 0.057 1

[0365] (2) Production of L-proline-3-hydroxylase by Transformant HavingL-proline-3-hydroxylase II-type Gene:

[0366]E. coli ATCC12435 was transformed with plasmid pEX1-5 as obtainedin Example 10. The resulting transformant was inoculated in 3 ml of a TBmedium containing 50 μg/ml of ampicillin and cultivated thereinovernight at 30° C. by shaking. The resulting culture was centrifuged,and the cells were collected. If desired, the cells were frozen andstored at −20° C., and the frozen cells were thawed before use.

[0367] The cells were added to 500 μl of a reaction mixture [containing25 mM L-proline, 50 mM 2-ketoglutaric acid, 4 mM ferrous sulfate and 8mM L-ascorbic acid in 200 mM TES buffer (pH 7.5)] at 2.2% (w/v) in termsof the wet cells, and reacted for 15 minutes at 35° C. The reactionmixture was heated at 100° C. for 2 minutes to stop the reaction.

[0368] The resulting reaction mixture was centrifuged, and 100 μl of 0.3M borate buffer (pH 10.7), 4 μl of 10% (v/v) mercaptoethanol aqueoussolution and 16 μl of 5% (w/v) OPA in ethanol were added to 100 μl ofthe resulting supernatant and left at 60° C. for 30 seconds, andthereafter the mixture was neutralized. This treatment resulted inmodification of primary amino acids with OPA but not the secondary aminoacid, hydroxyproline. The thus-processed reaction mixture was analyzedthrough HPLC, by which the product of cis-3-hydroxy-L-proline producedwas quantitatively determined. HPLC was conducted under the followingconditions.

[0369] Mobile Phase: aqueous solution of 1 mM copper sulfate.

[0370] Flow Rate: 1 ml/min.

[0371] Column: SUMICHIRAL OA-5000 (produced by Sumika Chemical AnalysisService Co., 4.6×250 mm)

[0372] Column Temperature: 38° C.

[0373] The eluate as optically resolved under the conditions mentionedabove was on-line mixed with reactants mentioned below under theconditions mentioned below, in which the eluate was modified with NBD ina reaction box CRB-6A (produced by Shimadzu Co.) and theNBDated-secondary amino acid was quantitatively determined throughfluorophotometry.

[0374] Reactants and Amounts Thereof Added:

[0375] aqueous solution of 300 mM boric acid and 25 mM EDTA (pH 9.6) 0.2ml/min.

[0376] 1 g/liter methanol of NBD: 0.5 ml/min.

[0377] Reaction Temperature: 60° C.

[0378] Detection: fluorophotometry

[0379] excitation wavelength 503 nm

[0380] emission wavelength 541 nm

[0381] As is shown in Table 14 below, the transformant E. coliATCC12435/pEX1-5 produced L-proline-3-hydroxylase more by approximately77 times/cell than the Streptomyces sp. TH1 strain which had been usedas the gene source. TABLE 14 L-proline-3-hydroxylase Activity Producedby Transformant Cell Relative Strain Activity¹⁾ Activity²⁾ E. coliATCC12435/pEX1-5 4.42 77.5 E. coli ATCC12435/pBluescriptII Not — KS(+)detected. Streptomyces sp. TH1³⁾ 0.057 1

EXAMPLE 12

[0382] Construction of Expression Plasmid for a Fused Protein with aβ-galactosidase Protein Fragment:

[0383] (1) Construction of pTH60 Plasmid:

[0384] Four micrograms of pTH40 DNA was cleaved with MluI and subjectedto ethanol precipitation to obtain an ethanol precipitate. Thethus-obtained ethanol precipitate (DNA fragment) was dissolved in 36 μlof TE, and the both terminals of the DNA fragment were blunted usingTakara DNA Blunting Kit (produced by Takara Shuzo Co., LTD). Theresulting DNA fragment was recovered through ethanol precipitation. TheDNA was cleaved with EcoRI and subjected to agarose gel electrophoresis,a fragment of approximately 1 kb containing the structural gene ofL-proline-3-hydroxylase was extracted from the agarose gel and recoveredin a usual manner using Pre-A-Gene (produced by Biorad Co.). The DNAfragment was then dissolved in 10 μl of TE.

[0385] On the other hand, 2.4 μg of plasmid pBluescriptII KS(+) DNA wascleaved with ApaI and subjected to agarose gel electrophoresis. The DNAfragment was then recovered from the gel and purified in a usual manner.Using Takara DNA Blunting Kit (produced by Takara Shuzo Co., LTD), theboth terminals of the DNA fragment were blunted. The thus-blunted DNAfragment was cleaved with EcoRI and then subjected to ethanolprecipitation to obtain an ethanol precipitate. The ethanol precipitatewas dissolved in 5 μl of TE.

[0386] The above-obtained MluI-EcoRI fragment of approximately 1 kbcontaining the structural gene of L-proline-3-hydroxylase was ligated tothe ApaI-EcoRI-cleaved pBluescriptII KS(+), using a ligation kit(produced by Takara Shuzo Co., LTD).

[0387] With the ligated DNA, E. coli XL1-Blue MRF′ strain wastransformed in a usual manner. The resulting transformant was spread onLB-agar medium containing 50 μg/ml of ampicillin and then cultivatedthereon overnight at 37° C. A plasmid was extracted from the growncolonies of the transformant in a usual manner, and its structure wasidentified through digestion with restriction enzyme.

[0388] As a result of the above, obtained was plasmid pTH60, into whichhad been inserted the structural gene of L-proline-3-hydroxylase asfused with the N-terminal amino acid sequence of β-Gal, in the directionsame as that for transcription of lac promoter (see FIG. 5). Thestructure of the amino acid sequence of the fused protein thusconstructed hereinabove was such that the N-terminal sequence of β-Galcomprised of 21 amino acids has been fused to theL-proline-3-hydroxylase protein via arginine (22nd) as newly formed atthe DNA bonding site. However, methionine (GTG) which is the first aminoacid of the L-proline-3-hydroxylase protein is translated as valine. Theamino acid sequence of the fused protein is indicated by Sequence No.15.

[0389] (2) pTH70 Plasmid:

[0390] pBluescriptII KS(+) DNA (2.4 μg) was cleaved with AccI andsubjected to ethanol precipitation to obtain an ethanol precipitate (DNAfragment). The both terminals of the DNA fragment were blunted usingTakara DNA Blunting Kit (produced by Takara Shuzo Co., LTD). Theresulting DNA fragment was cleaved with EcoRI and then subjected toethanol precipitation to obtain an ethanol precipitate. The ethanolprecipitate was dissolved in 5 μl of TE.

[0391] The MluI-EcoRI fragment of approximately 1 kb containing thestructural gene of L-proline-3-hydroxylase, which had been obtained in(1) of Example 12, was ligated to the AccI-EcoRI-cleaved pBluescriptIIKS (+), using a ligation kit (produced by Takara Shuzo Co., LTD).

[0392] With the ligated DNA, E. coli XL1-Blue MRF′ strain wastransformed in a usual manner. The resulting transformant was spread onLB-agar medium containing 50 μg/ml of ampicillin and then cultivatedthereon overnight at 37° C. A plasmid was extracted from the growncolonies of the transformant in a usual manner, and its structure wasidentified through digestion with restriction enzyme.

[0393] As a result of the above, obtained was plasmid pTH70, into whichhad been inserted the structural gene of L-proline-3-hydroxylase asfused with the N-terminal amino acid sequence of β-Gal in the directionsame as that for transcription of lac promoter (see FIG. 6). Thestructure of the amino acid sequence of the fused protein thusconstructed hereinabove was such that the N-terminal sequence of β-Galcomprised of 27 amino acids has been fused to theL-proline-3-hydroxylase via arginine (28th) as newly formed at the DNAbonding site. However, methionine (GTG) which is the first amino acid ofthe L-proline-3-hydroxylase protein is translated as valine. The aminoacid sequence of the fused protein is indicated by Sequence No. 16.

[0394] (3) pTH80 Plasmid:

[0395] Four mirograms of pTH40 DNA was cleaved with MluI and subjectedto ethanol precipitation to obtain an ethanol precipitate. The ethanolprecipitate (DNA fragment) was dissolved in 36 μl of TE. The bothterminals of the DNA fragment were blunted using Takara DNA Blunting Kit(produced by Takara Shuzo Co., LTD). The resulting DNA fragment cleavedwith SacII and then subjected to agarose gel electrophoresis. After theelectrophoresis, a fragment of approximately 0.95 kb containing thestructural gene of L-proline-3-hydroxylase was extracted from the geland recovered in a usual manner using Prep-A-Gene (produced by BioradCo.). The thus-recovered fragment was then dissolved in 10 μl of TE.

[0396] On the other hand, 2.4 μg of plasmid pBluescriptII KS(+) DNA wascleaved with PstI and subjected to ethanol precipitation to obtain anethanol precipitate (DNA fragment). This DNA fragment was dissolved in36 μl of TE, and the both terminals of the DNA fragment were bluntedusing Takara DNA Blunting Kit (produced by Takara Shuzo Co., LTD). Theresulting DNA fragment was then cleaved with SacII and subjected toethanol precipitation to obtain an ethanol precipitate (DNA fragment).The DNA fragment was then dissolved in 5 μl of TE.

[0397] The MluI-SacII fragment of approximately 1 kb containing thestructural gene of L-proline-3-hydroxylase, which had been obtained inthe above, was ligated to the PstI-SacII-cleaved pBluescriptII KS (+),using a ligation kit (produced by Takara Shuzo Co., LTD).

[0398] With the ligated DNA, E. coli XL1-Blue MRF′ strain wastransformed in a usual manner. The resulting transformant was spread onLB-agar medium containing 50 μg/ml of ampicillin and then cultivatedthereon overnight at 37° C. A plasmid was extracted from the growncolonies of the transformant in a usual manner, and its structure wasidentified through digestion with restriction enzyme.

[0399] As a result of the above, obtained was plasmid pTH80, into whichhad been inserted the structural gene of L-proline-3-hydroxylase asfused with the N-terminal amino acid sequence of 1-Gal in the directionsame as that for transcription of lac promoter (see FIG. 7). Thestructure of the amino acid sequence of the fused protein thusconstructed hereinabove was such that the N-terminal sequence of β-Galcomprised of 37 amino acids has been fused to theL-proline-3-hydroxylase protein via arginine (38th) as newly formed atthe DNA bonding site. However, methionine (GTG) which is the first aminoacid of the L-proline-3-hydroxylase protein is translated as valine. Theamino acid sequence of the fused protein is indicated by Sequence No.17.

EXAMPLE 13

[0400] Production of L-proline-3-hydroxylase by Transformant havingFused Protein Expression Plasmid:

[0401]E coli ATCC12435 was transformed with any one of plasmids pTH60,pTH70 and pTH80 as obtained in Example 12. In the same manner as inExample 11, the resulting transformant was cultivated, and theproductivity of L-proline-3-hydroxylase by the transformant cells wasdetected.

[0402] As shown in Table 15 below, the transformant producedL-proline-3-hydroxylase in an amount of 46 to 121 times per cell incomparison to Streptomyces sp. TH1 strain which had been used as thegene source. TABLE 15 L-proline-3-hydroxylase Activity Produced byTransformants Cell Relative Strain Activity¹⁾ Activity²⁾ E. coliATCC12435/pTH60 5.19 91.1 E. coli ATCC12435/pTH70 6.90 121.1 E. coliATCC12435/pTH80 2.63 46.1 E. coli ATCC12435/pBluescriptII Not — KS(+)detected. Streptomyces sp. TH1³⁾ 0.057 1.0

EXAMPLE 14

[0403] Production of Cis-3-hydroxy-L-proline by Transformant:

[0404] (1) Production of Cis-3-hydroxy-L-proline by Transformant E. coliATCC12435/pTH70:

[0405] Cis-3-hydroxy-L-proline was produced using the transformant E.coli ATCC12435/pTH70 as obtained in Example 13.

[0406] Precisely, the transformant of E. coli ATCC12435/pTH70 wasinoculated in 3 ml of a TB medium (comprising 12 g of bactotripton, 24 gof bactoyeast extract, 4 g of glycerol, 2.3 g of KH₂PO₄ and 12.5 g ofdipotassium phosphate in one liter of distilled water and sterilized at120° C. for 20 minutes) containing 100 μg/ml of ampicillin, andcultivated therein at 30° C. for 16 hours by shaking culture. Theresulting culture was centrifuged, and the amount ofcis-3-hydroxy-L-proline in the supernatant thus separated wasquantitatively determined.

[0407] As a result, 620 μM (81.1 mg/liter) of cis-3-hydroxy-L-prolinewas formed in the supernatant of the culture of E. coli ATCC12435/pTH70.

[0408] On the other hand, free cis-3-hydroxy-L-proline was not detectedin the supernatant of the culture of the E. coli ATCC12435 which hadbeen used as the host.

[0409] (2) Production of Cis-3-hydroxy-L-proline by Transformant E. coliATCC12435/pTH70:

[0410] The transformant of E. coli ATCC12435/pTH70 obtained in Example13 was inoculated in 50 ml of a Med4 medium containing 100 μg/ml ofampicillin, and cultivated therein at 30° C. for 16 hours by shaking.

[0411] The culture was used as a seed culture, which was inoculated in a5-liter jar fermenter containing therein 2 liters of a Med6 medium. 200mM of L-proline and 100 μg/ml of ampicillin were added thereto, and thetransformant was cultivated in the fermenter under the condition of 400rpm and 1 vvm, at 30° C.

[0412] During the cultivation, glucose and L-proline were suitably addedto the medium in such a manner that glucose was always present in themedium and L-proline could be at about 50 mM therein, and the lowermostlimit of the pH of the medium was controlled at 6.5 by adding NH₄OH tothe medium.

[0413] The culture was centrifuged, and the amount ofcis-3-hydroxy-L-proline in the supernatant separated was quantitativelydetermined. Seventy two hours after the start of the incubation, 115 mM(15 g/liter) of cis-3-hydroxy-L-proline was produced and accumulated inthe supernatant of the culture of E. coli ATCC12435/pTH70.

[0414] On the other hand, free cis-3-hydroxy-L-proline was not detectedin the supernatant of the culture of E. coli ATCC12435 which had beenused as the host.

EXAMPLE 15

[0415] Conversion of L-proline into Cis-3-hydroxy-L-Proline withTransformant Cells:

[0416] Using the transformant E. coli ATCC12435/pTH70 as obtained inExample 13, L-proline was converted into cis-3-hydroxy-L-proline.

[0417] Precisely, the transformant was inoculated in 10 ml of an LBmedium containing 50 μg/ml of ampicillin and cultivated thereinovernight at 30° C. by shaking. Then 7 ml of the culture was centrifugedto collect the cells (wet cells). If desired, the cells were frozen andstored at −20° C. and thawed before use.

[0418] After 10%(w/v) of the wet cells were added to 500 μl of areaction mixture [comprising 24 mM L-proline, 24 mM 2-ketoglutaric acid,4 mM ferrous sulfate and 8 mM L-ascorbic acid in 200 mM TES buffer (pH7.5)], reaction was conducted at 35° C. for 60 minutes. The amount ofcis-3-hydroxy-L-proline as formed in the reaction mixture wasquantitatively determined. As a result, 17.7 mM (2.3 g/liter) ofcis-3-hydroxy-L-proline was formed in the reaction mixture.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:17 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 290 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptomycessp. (B) STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Arg SerHis Ile Leu Gly Arg Ile Glu Leu Asp Gln Glu Arg Leu 1 5 10 15 Gly ArgAsp Leu Glu Tyr Leu Ala Thr Val Pro Thr Val Glu Glu Glu 20 25 30 Tyr AspGlu Phe Ser Asn Gly Phe Trp Lys Asn Ile Pro Leu Tyr Asn 35 40 45 Ala SerGly Gly Ser Glu Asp Arg Leu Tyr Arg Asp Leu Glu Gly Ser 50 55 60 Pro AlaGln Pro Thr Lys His Ala Glu Gln Val Pro Tyr Leu Asn Glu 65 70 75 80 IleIle Thr Thr Val Tyr Asn Gly Glu Arg Leu Gln Met Ala Arg Thr 85 90 95 ArgAsn Leu Lys Asn Ala Val Val Ile Pro His Arg Asp Phe Val Glu 100 105 110Leu Asp Arg Glu Leu Asp Gln Tyr Phe Arg Thr His Leu Met Leu Glu 115 120125 Asp Ser Pro Leu Ala Phe His Ser Asp Asp Asp Thr Val Ile His Met 130135 140 Arg Ala Gly Glu Ile Trp Phe Leu Asp Ala Ala Ala Val His Ser Ala145 150 155 160 Val Asn Phe Ala Glu Phe Ser Arg Gln Ser Leu Cys Val AspLeu Ala 165 170 175 Phe Asp Gly Ala Phe Asp Glu Lys Glu Ala Phe Ala AspAla Thr Val 180 185 190 Tyr Ala Pro Asn Leu Ser Pro Asp Val Arg Glu ArgLys Pro Phe Thr 195 200 205 Lys Glu Arg Glu Ala Gly Ile Leu Ala Leu SerGly Val Ile Gly Arg 210 215 220 Glu Asn Phe Arg Asp Ile Leu Phe Leu LeuSer Lys Val His Tyr Thr 225 230 235 240 Tyr Asp Val His Pro Gly Glu ThrPhe Glu Trp Leu Val Ser Val Ser 245 250 255 Lys Gly Ala Gly Asp Asp LysMet Val Glu Lys Ala Glu Arg Ile Arg 260 265 270 Asp Phe Ala Ile Gly AlaArg Ala Leu Gly Glu Arg Phe Ser Leu Thr 275 280 285 Thr Trp 290 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:290 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptomyces sp. (B)STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Arg Ser His IleLeu Gly Lys Ile Glu Leu Asp Gln Thr Arg Leu 1 5 10 15 Ala Pro Asp LeuAla Tyr Leu Ala Ala Val Pro Thr Val Glu Glu Glu 20 25 30 Tyr Asp Glu PheSer Asn Gly Phe Trp Lys His Val Pro Leu Trp Asn 35 40 45 Ala Ser Gly AspSer Glu Asp Arg Leu Tyr Arg Asp Leu Lys Asp Ala 50 55 60 Ala Ala Gln ProThr Ala His Val Glu His Val Pro Tyr Leu Lys Glu 65 70 75 80 Ile Val ThrThr Val Phe Asp Gly Thr His Leu Gln Met Ala Arg Ser 85 90 95 Arg Asn LeuLys Asn Ala Ile Val Ile Pro His Arg Asp Phe Val Glu 100 105 110 Leu AspArg Glu Val Asp Arg Tyr Phe Arg Thr Phe Met Val Leu Glu 115 120 125 AspSer Pro Leu Ala Phe His Ser Asn Glu Asp Thr Val Ile His Met 130 135 140Arg Pro Gly Glu Ile Trp Phe Leu Asp Ala Ala Thr Val His Ser Ala 145 150155 160 Val Asn Phe Ser Glu Ile Ser Arg Gln Ser Leu Cys Val Asp Phe Ala165 170 175 Phe Asp Gly Pro Phe Asp Glu Lys Glu Ile Phe Ala Asp Ala ThrLeu 180 185 190 Tyr Ala Pro Gly Ser Thr Pro Asp Leu Pro Glu Arg Arg ProPhe Thr 195 200 205 Ala Glu His Arg Arg Arg Ile Leu Ser Leu Gly Gln ValIle Glu Arg 210 215 220 Glu Asn Phe Arg Asp Ile Leu Phe Leu Leu Ser LysVal His Tyr Lys 225 230 235 240 Tyr Asp Val His Pro Ser Glu Thr Tyr AspTrp Leu Ile Glu Ile Ser 245 250 255 Lys Gln Ala Gly Asp Glu Lys Met ValVal Lys Ala Glu Gln Ile Arg 260 265 270 Asp Phe Ala Val Glu Ala Arg AlaLeu Ser Glu Arg Phe Ser Leu Thr 275 280 285 Ser Trp 290 (2) INFORMATIONFOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 870 basepaires (B) TYPE: nucleic acid (C) STRANDEDNESS: double (ii) MOLECULETYPE: genomic DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptomyces sp.(B) STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTGCGCTCGCACATACTCGG CCGCATTGAA CTCGACCAGG AACGCTTAGG CAGGGACCTC 60 GAATATCTCGCCACGGTGCC CACCGTGGAA GAGGAGTACG ACGAGTTCAG CAACGGGTTC 120 TGGAAGAACATCCCGCTGTA CAACGCGAGC GGCGGCAGCG AGGACCGGCT GTACCGCGAC 180 CTCGAGGGCTCACCGGCGCA GCCCACCAAA CACGCCGAGC AGGTTCCGTA CCTCAACGAG 240 ATCATCACCACGGTCTACAA CGGCGAGCGG CTCCAGATGG CGCGTACGCG GAACCTGAAG 300 AACGCCGTCGTCATCCCGCA CCGCGACTTC GTGGAGCTCG ACCGCGAACT CGACCAGTAC 360 TTCCGCACCCATTTGATGCT TGAGGACAGC CCGCTGGCCT TCCACTCGGA CGACGACACC 420 GTCATCCACATGCGGGCCGG CGAGATCTGG TTCCTCGACG CGGCCGCCGT CCACTCGGCC 480 GTCAACTTCGCCGAGTTCAG CAGGCAGTCG CTCTGCGTCG ACCTCGCCTT CGACGGCGCG 540 TTCGACGAGAAGGAAGCCTT CGCGGACGCC ACGGTCTACG CCCCGAACCT CAGCCCCGAC 600 GTCCGCGAACGCAAGCCGTT CACCAAGGAG CGGGAGGCCG GGATCCTCGC CCTGTCCGGC 660 GTGATCGGACGCGAGAACTT CCGGGACATC CTCTTTCTGC TGTCCAAGGT CCACTACACC 720 TACGACGTCCATCCGGGTGA AACCTTCGAG TGGCTCGTGA GCGTCTCCAA GGGTGCGGGA 780 GACGACAAGATGGTGGAGAA GGCCGAGCGG ATCAGGGACT TCGCCATCGG CGCACGGGCA 840 CTCGGCGAGCGTTTCTCGCT GACCACCTGG 870 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 870 base paires (B) TYPE: nucleic acid (C)STRANDEDNESS: double (ii) MOLECULE TYPE: genomic DNA (vi) ORIGINALSOURCE: (A) ORGANISM: Streptomyces sp. (B) STRAIN: TH1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: ATGCGCTCGC ACATCCTTGG CAAGATCGAA CTCGATCAGACCCGGTTGGC GCCGGATCTC 60 GCATACCTCG CCGCAGTTCC GACCGTGGAG GAGGAGTACGACGAGTTCAG CAACGGGTTC 120 TGGAAGCACG TGCCCCTGTG GAACGCCTCC GGGGACAGCGAGGACCGGCT CTACCGCGAC 180 CTCAAGGACG CCGCCGCACA GCCGACCGCG CACGTGGAGCACGTCCCCTA CCTCAAGGAG 240 ATCGTGACCA CGGTCTTCGA CGGCACGCAC CTGCAGATGGCGCGGAGCCG GAACCTGAAG 300 AACGCCATCG TCATCCCGCA CCGCGACTTC GTGGAGCTGGACCGCGAAGT CGACCGGTAC 360 TTCCGCACGT TCATGGTGCT GGAGGACAGC CCGCTCGCCTTCCACTCGAA CGAGGACACC 420 GTCATCCACA TGCGCCCGGG CGAAATATGG TTCCTGGACGCGGCGACGGT GCACTCCGCG 480 GTCAACTTCT CGGAAATCAG CCGTCAGTCC CTGTGCGTCGACTTCGCCTT CGACGGTCCC 540 TTCGACGAGA AGGAGATCTT CGCGGACGCC ACCCTCTACGCTCCGGGCTC CACGCCCGAC 600 CTGCCCGAGC GCCGCCCCTT CACCGCGGAG CACCGGCGGCGGATCCTCTC CCTGGGCCAG 660 GTGATCGAGC GGGAGAACTT CCGGGACATT CTGTTCCTGCTGTCCAAGGT GCACTACAAG 720 TACGACGTGC ACCCCAGCGA GACGTACGAC TGGCTGATCGAGATCTCGAA ACAGGCCGGC 780 GACGAGAAGA TGGTCGTGAA GGCGGAGCAG ATCAGGGACTTCGCCGTCGA GGCCCGCGCC 840 CTGAGCGAGC GCTTCTCCCT GACCTCCTGG 870 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (v) FRAGMENT TYPE: N-terminal fragment (vi) ORIGINALSOURCE: (A) ORGANISM: Streptomyces sp. (B) STRAIN: TH1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: Met Arg Ser His Ile Leu Gly Arg Ile Glu LeuAsp Gln Glu Arg Leu 1 5 10 15 Gly Arg Asp Leu Glu Tyr Leu Ala Thr ValPro Thr Val 20 25 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internalfragment (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptomyces sp. (B)STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Glu Ala Phe Ala AspAla Thr Val Tyr Ala Pro Asn Leu Ser Pro Asp 1 5 10 15 Val Arg Glu ArgLys Pro Phe Thr Lys 20 25 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internalfragment (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptomyces sp. (B)STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Asn Ile Pro Leu TyrAsn Ala Ser Gly Gly Ser Glu Asp Arg Leu Tyr 1 5 10 15 Arg Asp Leu GluGly Ser Pro Ala Gln 20 25 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base paires (B) TYPE: nucleic acid (C)STRANDEDNESS: single (ii) MOLECULE TYPE: other mucleic acid, syntheticDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGTGYTCNC AYATHYT 17 (2)INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 base paires (B) TYPE: nucleic acid (C) STRANDEDNESS: single (ii)MOLECULE TYPE: other mucleic acid, synthetic DNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 9: GTNGGNACNG TWGC 14 (2) INFORMATION FOR SEQ IDNO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base paires (B)TYPE: nucleic acid (C) STRANDEDNESS: single (ii) MOLECULE TYPE: othermucleic acid, synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:GTNGCNAGRT AYTC 14 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 74 base paires (B) TYPE: nucleic acid (C)STRANDEDNESS: double (ii) MOLECULE TYPE: other nucleic acid, syntheticDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG TGT TCG CAT ATA CTCGGC CGC ATT GAA CTC GAC CAG GAA CGC TTA 48 Met Cys Ser His Ile Leu GlyArg Ile Glu Leu Asp Gln Glu Arg Leu 1 5 10 15 GGC AGG GAC CTC GAA TACCTC GCC AC 74 Gly Arg Asp Leu Glu Tyr Leu Ala 20 (2) INFORMATION FOR SEQID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 base paires (B)TYPE: nucleic acid (C) STRANDEDNESS: double (ii) MOLECULE TYPE: othernucleic acid, synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:ATG TGC TCG CAT ATC CTT GGC AAG ATC GAA CTC GAT CAG ACC CGG TTG 48 MetCys Ser His Ile Leu Gly Lys Ile Glu Leu Asp Gln Thr Arg Leu 1 5 10 15GCG CCG GAT CTC GAG TAC CTC GCC AC 74 Ala Pro Asp Leu Glu Tyr Leu Ala 20(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1081 base paires (B) TYPE: nucleic acid (C) STRANDEDNESS: double(ii) MOLECULE TYPE: genomic DNA (vi) ORIGINAL SOURCE: (A) ORGANISM:Streptomyces sp. (B) STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGTACCCATT CCGTCACCGA AAGCTGAACT CGACACCAGG AGGACGACGC GTGCGCTCGC 60ACATACTCGG CCGCATTGAA CTCGACCAGG AACGCTTAGG CAGGGACCTC GAATATCTCG 120CCACGGTGCC CACCGTGGAA GAGGAGTACG ACGAGTTCAG CAACGGGTTC TGGAAGAACA 180TCCCGCTGTA CAACGCGAGC GGCGGCAGCG AGGACCGGCT GTACCGCGAC CTCGAGGGCT 240CACCGGCGCA GCCCACCAAA CACGCCGAGC AGGTTCCGTA CCTCAACGAG ATCATCACCA 300CGGTCTACAA CGGCGAGCGG CTCCAGATGG CGCGTACGCG GAACCTGAAG AACGCCGTCG 360TCATCCCGCA CCGCGACTTC GTGGAGCTCG ACCGCGAACT CGACCAGTAC TTCCGCACCC 420ATTTGATGCT TGAGGACAGC CCGCTGGCCT TCCACTCGGA CGACGACACC GTCATCCACA 480TGCGGGCCGG CGAGATCTGG TTCCTCGACG CGGCCGCCGT CCACTCGGCC GTCAACTTCG 540CCGAGTTCAG CAGGCAGTCG CTCTGCGTCG ACCTCGCCTT CGACGGCGCG TTCGACGAGA 600AGGAAGCCTT CGCGGACGCC ACGGTCTACG CCCCGAACCT CAGCCCCGAC GTCCGCGAAC 660GCAAGCCGTT CACCAAGGAG CGGGAGGCCG GGATCCTCGC CCTGTCCGGC GTGATCGGAC 720GCGAGAACTT CCGGGACATC CTCTTTCTGC TGTCCAAGGT CCACTACACC TACGACGTCC 780ATCCGGGTGA AACCTTCGAG TGGCTCGTGA GCGTCTCCAA GGGTGCGGGA GACGACAAGA 840TGGTGGAGAA GGCCGAGCGG ATCAGGGACT TCGCCATCGG CGCACGGGCA CTCGGCGAGC 900GTTTCTCGCT GACCACCTGG TAGACGGCCG AAGAACGGGG CCCGGGGGAA CAGTGATCGA 960GGAGATACTT CCCGTCGACG TGATGTCCGC GGAGGCGTTC GACGACGATG CGGACATCCA 1020GCTCTTCGCC GAGGAACGCG CGGCCGTCGC CGATGCCGTA CCGCGGCGCC GACGGGAATT 1080 C(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1826 base paires (B) TYPE: nucleic acid (C) STRANDEDNESS: double(ii) MOLECULE TYPE: genomic DNA (vi) ORIGINAL SOURCE: (A) ORGANISM:Streptomyces sp. (B) STRAIN: TH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TCGCGACCTG GAGGGTCGGA TGCGCTCGCA CATCCTTGGC AAGATCGAAC TCGATCAGAC 60CCGGTTGGCG CCGGATCTCG CATACCTCGC CGCAGTTCCG ACCGTGGAGG AGGAGTACGA 120CGAGTTCAGC AACGGGTTCT GGAAGCACGT GCCCCTGTGG AACGCCTCCG GGGACAGCGA 180GGACCGGCTC TACCGCGACC TCAAGGACGC CGCCGCACAG CCGACCGCGC ACGTGGAGCA 240CGTCCCCTAC CTCAAGGAGA TCGTGACCAC GGTCTTCGAC GGCACGCACC TGCAGATGGC 300GCGGAGCCGG AACCTGAAGA ACGCCATCGT CATCCCGCAC CGCGACTTCG TGGAGCTGGA 360CCGCGAAGTC GACCGGTACT TCCGCACGTT CATGGTGCTG GAGGACAGCC CGCTCGCCTT 420CCACTCGAAC GAGGACACCG TCATCCACAT GCGCCCGGGC GAAATATGGT TCCTGGACGC 480GGCGACGGTG CACTCCGCGG TCAACTTCTC GGAAATCAGC CGTCAGTCCC TGTGCGTCGA 540CTTCGCCTTC GACGGTCCCT TCGACGAGAA GGAGATCTTC GCGGACGCCA CCCTCTACGC 600TCCGGGCTCC ACGCCCGACC TGCCCGAGCG CCGCCCCTTC ACCGCGGAGC ACCGGCGGCG 660GATCCTCTCC CTGGGCCAGG TGATCGAGCG GGAGAACTTC CGGGACATTC TGTTCCTGCT 720GTCCAAGGTG CACTACAAGT ACGACGTGCA CCCCAGCGAG ACGTACGACT GGCTGATCGA 780GATCTCGAAA CAGGCCGGCG ACGAGAAGAT GGTCGTGAAG GCGGAGCAGA TCAGGGACTT 840CGCCGTCGAG GCCCGCGCCC TGAGCGAGCG CTTCTCCCTG ACCTCCTGGT AATGGCGCGA 900CCTCAGCCCG CGTGACAACA GTTCGGCCCC GGTGGCGCGG NGCGTCCCGG ACGCGCGCCG 960GNCGCTACCG GACACCGGGG CACGTAGGTG GTGTACCGNC TGNCGGTCTC CGGCAGTCAG 1020TTGCTGGTTC GACTCCAGCT GCCCCGACCG CCTCGCGCTC GGACGCGACC CCGGGCACCT 1080CCGNGGGCAC CGCGTTCGCA TCACGGACAC CCCACCCACA CCAGCCGGGC CCACCCGAGG 1140AGCCAGTTCA CATGTCCAGT GACATACACG CGGCGGCATC CAGACCGATT CCGCCGAAGC 1200CCGCCGCATC GACGGAGGTC GCGCCGCGCA CGCTGGTCAG CCGGCTGCCC TCGCTGACCG 1260GCCTGCGCTT CCCGGCGGCG TTCATCGTGT TCCTCTTCCA CGCCTCGCTG CCGTTTCCCG 1320CGGTGCGCCT GTTCGCCGAC GACGGGGTGG AACACCGCTA CGGGTGGGCC CTCGGCCCGA 1380GCGGCGCACT CGGCGTGACG TTCTTCTTCG CGCTCAGCGG ATTCGTGCTG ACGTGGTCGG 1440CTCCCGCCGG CGACACCGCA CCGTCCTTCT GGCGCCGGCG CTTCGTCAAG ATCCTGCCCA 1500ACTACGTCGT CGCGTGGGTC CTGGCGATGG TGCTGTACGC GGCCGCGACG CCCGTCCTGC 1560CCGCGCTCGG CGCCCTTTTC ATGCTCCAGG TGTGGACGCC GTACTTCACC GAGCACCTCC 1620CGGTGAACCC ACCGAGCTGG TCGCTGNCCG TGGAAGCCGT CTTCTATCTG GCCTTCCCGT 1680TCCTGCTGGC CGGGATCAGA CGGATACCGG CCGCCCGGCT GAAGTACTGG ATCGCCGGCA 1740CGGTGGCCGC CGTCTTCGCC ACGCCGCTGA TCACCTACCT CCTGGTGCCG GCGGGCCCGC 1800ACGTGATGCC GGGCACGGGC GGTACC 1826 (2) INFORMATION FOR SEQ ID NO: 15: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 312 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINALSOURCE: (A) ORGANISM: Escherichia coli (B) STRAIN: TH1 (vii) IMMEDIATESOURCE: (ix) FEATURE: (A) NAME/KEY: peptide (B) LOCATION: 23 to 312 (C)IDENTIFICATION METHOD: by similarity with known sequence or to anestablished consensus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Met ThrMet Ile Thr Pro Ser Ala Gln Leu Thr Leu Thr Lys Gly Asn 1 5 10 15 LysSer Trp Val Pro Arg Val Arg Ser His Ile Leu Gly Arg Ile Glu 20 25 30 LeuAsp Gln Glu Arg Leu Gly Arg Asp Leu Glu Tyr Leu Ala Thr Val 35 40 45 ProThr Val Glu Glu Glu Tyr Asp Glu Phe Ser Asn Gly Phe Trp Lys 50 55 60 AsnIle Pro Leu Tyr Asn Ala Ser Gly Gly Ser Glu Asp Arg Leu Tyr 65 70 75 80Arg Asp Leu Glu Gly Ser Pro Ala Gln Pro Thr Lys His Ala Glu Gln 85 90 95Val Pro Tyr Leu Asn Glu Ile Ile Thr Thr Val Tyr Asn Gly Glu Arg 100 105110 Leu Gln Met Ala Arg Thr Arg Asn Leu Lys Asn Ala Val Val Ile Pro 115120 125 His Arg Asp Phe Val Glu Leu Asp Arg Glu Leu Asp Gln Tyr Phe Arg130 135 140 Thr His Leu Met Leu Glu Asp Ser Pro Leu Ala Phe His Ser AspAsp 145 150 155 160 Asp Thr Val Ile His Met Arg Ala Gly Glu Ile Trp PheLeu Asp Ala 165 170 175 Ala Ala Val His Ser Ala Val Asn Phe Ala Glu PheSer Arg Gln Ser 180 185 190 Leu Cys Val Asp Leu Ala Phe Asp Gly Ala PheAsp Glu Lys Glu Ala 195 200 205 Phe Ala Asp Ala Thr Val Tyr Ala Pro AsnLeu Ser Pro Asp Val Arg 210 215 220 Glu Arg Lys Pro Phe Thr Lys Glu ArgGlu Ala Gly Ile Leu Ala Leu 225 230 235 240 Ser Gly Val Ile Gly Arg GluAsn Phe Arg Asp Ile Leu Phe Leu Leu 245 250 255 Ser Lys Val His Tyr ThrTyr Asp Val His Pro Gly Glu Thr Phe Glu 260 265 270 Trp Leu Val Ser ValSer Lys Gly Ala Gly Asp Asp Lys Met Val Glu 275 280 285 Lys Ala Glu ArgIle Arg Asp Phe Ala Ile Gly Ala Arg Ala Leu Gly 290 295 300 Glu Arg PheSer Leu Thr Thr Trp 305 310 (2) INFORMATION FOR SEQ ID NO: 16: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINALSOURCE: (A) ORGANISM: Escherichia coli (B) STRAIN: TH1 (vii) IMMEDIATESOURCE: (ix) FEATURE: (A) NAME/KEY: peptide (B) LOCATION: 29 to 318 (C)IDENTIFICATION METHOD: by similarity with known sequence or to anestablished consensus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Met ThrMet Ile Thr Pro Ser Ala Gln Leu Thr Leu Thr Lys Gly Asn 1 5 10 15 LysSer Trp Val Pro Gly Pro Pro Ser Arg Ser Arg Val Arg Ser His 20 25 30 IleLeu Gly Arg Ile Glu Leu Asp Gln Glu Arg Leu Gly Arg Asp Leu 35 40 45 GluTyr Leu Ala Thr Val Pro Thr Val Glu Glu Glu Tyr Asp Glu Phe 50 55 60 SerAsn Gly Phe Trp Lys Asn Ile Pro Leu Tyr Asn Ala Ser Gly Gly 65 70 75 80Ser Glu Asp Arg Leu Tyr Arg Asp Leu Glu Gly Ser Pro Ala Gln Pro 85 90 95Thr Lys His Ala Glu Gln Val Pro Tyr Leu Asn Glu Ile Ile Thr Thr 100 105110 Val Tyr Asn Gly Glu Arg Leu Gln Met Ala Arg Thr Arg Asn Leu Lys 115120 125 Asn Ala Val Val Ile Pro His Arg Asp Phe Val Glu Leu Asp Arg Glu130 135 140 Leu Asp Gln Tyr Phe Arg Thr His Leu Met Leu Glu Asp Ser ProLeu 145 150 155 160 Ala Phe His Ser Asp Asp Asp Thr Val Ile His Met ArgAla Gly Glu 165 170 175 Ile Trp Phe Leu Asp Ala Ala Ala Val His Ser AlaVal Asn Phe Ala 180 185 190 Glu Phe Ser Arg Gln Ser Leu Cys Val Asp LeuAla Phe Asp Gly Ala 195 200 205 Phe Asp Glu Lys Glu Ala Phe Ala Asp AlaThr Val Tyr Ala Pro Asn 210 215 220 Leu Ser Pro Asp Val Arg Glu Arg LysPro Phe Thr Lys Glu Arg Glu 225 230 235 240 Ala Gly Ile Leu Ala Leu SerGly Val Ile Gly Arg Glu Asn Phe Arg 245 250 255 Asp Ile Leu Phe Leu LeuSer Lys Val His Tyr Thr Tyr Asp Val His 260 265 270 Pro Gly Glu Thr PheGlu Trp Leu Val Ser Val Ser Lys Gly Ala Gly 275 280 285 Asp Asp Lys MetVal Glu Lys Ala Glu Arg Ile Arg Asp Phe Ala Ile 290 295 300 Gly Ala ArgAla Leu Gly Glu Arg Phe Ser Leu Thr Thr Trp 305 310 315 (2) INFORMATIONFOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 328 aminoacids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (B) STRAIN:TH1 (vii) IMMEDIATE SOURCE: (ix) FEATURE: (A) NAME/KEY: peptide (B)LOCATION: 38 to 328 (C) IDENTIFICATION METHOD: by similarity with knownsequence or to an established consensus (xi) SEQUENCE DESCRIPTION: SEQID NO: 17: Met Thr Met Ile Thr Pro Ser Ala Gln Leu Thr Leu Thr Lys GlyAsn 1 5 10 15 Lys Ser Trp Val Pro Gly Pro Pro Ser Arg Ser Thr Val SerIle Ser 20 25 30 Leu Ile Ser Asn Ser Arg Val Arg Ser His Ile Leu Gly ArgIle Glu 35 40 45 Leu Asp Gln Glu Arg Leu Gly Arg Asp Leu Glu Tyr Leu AlaThr Val 50 55 60 Pro Thr Val Glu Glu Glu Tyr Asp Glu Phe Ser Asn Gly PheTrp Lys 65 70 75 80 Asn Ile Pro Leu Tyr Asn Ala Ser Gly Gly Ser Glu AspArg Leu Tyr 85 90 95 Arg Asp Leu Glu Gly Ser Pro Ala Gln Pro Thr Lys HisAla Glu Gln 100 105 110 Val Pro Tyr Leu Asn Glu Ile Ile Thr Thr Val TyrAsn Gly Glu Arg 115 120 125 Leu Gln Met Ala Arg Thr Arg Asn Leu Lys AsnAla Val Val Ile Pro 130 135 140 His Arg Asp Phe Val Glu Leu Asp Arg GluLeu Asp Gln Tyr Phe Arg 145 150 155 160 Thr His Leu Met Leu Glu Asp SerPro Leu Ala Phe His Ser Asp Asp 165 170 175 Asp Thr Val Ile His Met ArgAla Gly Glu Ile Trp Phe Leu Asp Ala 180 185 190 Ala Ala Val His Ser AlaVal Asn Phe Ala Glu Phe Ser Arg Gln Ser 195 200 205 Leu Cys Val Asp LeuAla Phe Asp Gly Ala Phe Asp Glu Lys Glu Ala 210 215 220 Phe Ala Asp AlaThr Val Tyr Ala Pro Asn Leu Ser Pro Asp Val Arg 225 230 235 240 Glu ArgLys Pro Phe Thr Lys Glu Arg Glu Ala Gly Ile Leu Ala Leu 245 250 255 SerGly Val Ile Gly Arg Glu Asn Phe Arg Asp Ile Leu Phe Leu Leu 260 265 270Ser Lys Val His Tyr Thr Tyr Asp Val His Pro Gly Glu Thr Phe Glu 275 280285 Trp Leu Val Ser Val Ser Lys Gly Ala Gly Asp Asp Lys Met Val Glu 290295 300 Lys Ala Glu Arg Ile Arg Asp Phe Ala Ile Gly Ala Arg Ala Leu Gly305 310 315 320 Glu Arg Phe Ser Leu Thr Thr Trp 325

What is claimed is:
 1. A microorganism having an enzymatic activity ofcatalyzing hydroxylation of L-proline at the 3-position of L-proline. 2.The microorganism according to claim 1, wherein the microorganismbelongs to the genus Streptomyces or Bacillus.
 3. A microorganism havingan ability to produce L-proline-3-hydroxylase.
 4. The microorganismaccording to claim 3, wherein the microorganism belongs to the genusStreptomyces or Bacillus.
 5. A microorganism selected from a groupconsisting of Streptomyces sp.TH1 (FERM BP-4399), Bacillus sp.TH2 (FERMBP-4397) and Bacillus sp.TH3 (FERM BP-4398)